WO2007007116A1 - Proteines de type defensine - Google Patents

Proteines de type defensine Download PDF

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Publication number
WO2007007116A1
WO2007007116A1 PCT/GB2006/002636 GB2006002636W WO2007007116A1 WO 2007007116 A1 WO2007007116 A1 WO 2007007116A1 GB 2006002636 W GB2006002636 W GB 2006002636W WO 2007007116 A1 WO2007007116 A1 WO 2007007116A1
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Prior art keywords
polypeptide
seq
nucleic acid
disease
acid molecule
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PCT/GB2006/002636
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English (en)
Inventor
Melanie Yorke
Christine Power
Jadwiga Bienkowska
David Michalovich
Simon John White
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Ares Trading S.A
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Application filed by Ares Trading S.A filed Critical Ares Trading S.A
Priority to EP06764978A priority Critical patent/EP1904523A1/fr
Publication of WO2007007116A1 publication Critical patent/WO2007007116A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins

Definitions

  • This invention relates to the novel protein, termed INSP 139, herein identified as a secreted protein, in particular, as a member of the defensin family of proteins and to the use of this protein and nucleic acid sequence from the encoding gene in the diagnosis, prevention and treatment of disease.
  • bioinformatics tools increase in potency and in accuracy, these tools are rapidly replacing the conventional techniques of biochemical characterisation. Indeed, the advanced bioinformatics tools used in identifying the present invention are now capable of outputting results in which a high degree of confidence can be placed.
  • Enzymes, growth factors, extracellular matrix proteins and signalling molecules are all secreted by cells. This is through fusion of a secretory vesicle with the plasma membrane. In most cases, but not all, proteins are directed to the endoplasmic reticulum and into secretory vesicles by a signal peptide.
  • Signal peptides are cis-acting sequences that affect the transport of polypeptide chains from the cytoplasm to a membrane bound compartment such as a secretory vesicle. Polypeptides that are targeted to the secretory vesicles are either secreted into the extracellular matrix or are retained in the plasma membrane.
  • polypeptides that are retained in the plasma membrane will have one or more transmembrane domains.
  • secreted proteins that play a central role in the functioning of a cell are cytokines, hormones, extracellular matrix proteins (adhesion molecules), proteases, and growth and differentiation factors. Description of some of the properties of these proteins follows.
  • Defensins form part of the body's innate immune system that acts against invasion by foreign pathogens. Mammalian defensins are split into three categories; alpha, beta and theta, based upon the pattern of disulphide bonds. INSP 139 falls into the beta category. These proteins are cationic and arginine-rich and share a typical tertiary structure despite differences in primary structure. They consist of three antiparallel beta sheets connected by loops, and a beta hairpin with hydrophobic properties protrudes orthogonally.
  • the invention is based on the discovery that the INSP 139 polypeptide is a beta defensin.
  • the INSP 139 polypeptide is a beta defensin.
  • (i) comprises the amino acid sequence as recited in SEQ E ) NO:2, SEQ ID NO:4, SEQ ID NO:6 and/or SEQ ID NO:8;
  • (ii) is a fragment thereof which functions as a member of the defensin family of proteins, or has an antigenic determinant in common with the polypeptides of (i); or
  • (i) comprises the amino acid sequence as recited in SEQ ID NO:6 and/or SEQ ID NO:8;
  • (ii) is a fragment thereof which functions as a member of the defensin family of proteins, or has an antigenic determinant in common with the polypeptides of (i); or
  • (i) consists of the amino acid sequence as recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and/or SEQ ID NO:8;
  • (ii) is a fragment thereof which functions as a member of the defensin family of proteins, or having an antigenic determinant in common with the polypeptides of (i); or
  • polypeptide having the sequence recited in SEQ ID NO:2 is referred to hereafter as "INSP 139 exon 1 polypeptide".
  • polypeptide having the sequence recited in SEQ ID NO:4 is referred to hereafter as "INSP139 exon 2 polypeptide”.
  • polypeptide having the sequence recited in SEQ ID NO:6 is referred to hereafter as the "INSP139 polypeptide”.
  • the full length INSP139 polypeptide sequence without this postulated signal sequence is recited in SEQ ID NO:8.
  • the polypeptide having the sequence recited in SEQ ID NO:8 is referred to hereafter as "the INSP139 mature polypeptide".
  • the present invention also includes a splice variant of the INSP 139 polypeptide sequence.
  • This splice variant hereafter called INSP 139-SVl 5 is recited in SEQ ID NO: 10.
  • INSP 139-SVl 5 is recited in SEQ ID NO: 10.
  • the first 22 amino acids of the INSP139-SV1 polypeptide form a signal peptide.
  • the full length INSP139-SV1 polypeptide sequence without this postulated signal sequence is recited in SEQ ID NO: 12.
  • the polypeptide having the sequence recited in SEQ ID NO: 12 is referred to hereafter as "the INSP139-SV1 mature polypeptide".
  • the invention also includes His-tagged INSP139 and INSP139-SV polypeptide sequences.
  • the polypeptide having the sequence recited in SEQ ID NO: 13 is referred to hereafter as "the INSP 139 His tag polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 14 is referred to hereafter as "the INSP139-SV1 His tag polypeptide”.
  • the INSP139 and INSP139-SV1 polypeptides have been discovered where residue 21 is G rather than R (for INSP139) or V (for INSP139-SV1).
  • These polypeptides are referred to hereafter as “the INSP139 R21G polypeptide” (SEQ ID NO: 15) and “the INSP139-SV1 V21G polypeptide” (SEQ ID NO: 16).
  • His tagged versions of these polypeptides are also included in the invention and these are referred to as “the INSP139 R21G His tag polypeptide” (SEQ ID NO: 17) and "the INSP139-SV1 V21G His tag polypeptide” (SEQ ID NO: 18).
  • ⁇ -defensin genes encode a precursor peptide that consists of a hydrophobic, leucine- rich signal sequence, a pro-sequence, and a mature six-cysteine defensin motif at the carboxy terminus, hi most cases, ⁇ -defensin precursors are encoded in two separate exons separated by an intron of variable length, with one exon encoding the signal and pro- sequence and the other encoding primarily the mature peptide. Patil et al.
  • comparison of the primary structures for ⁇ -defensins from various sources defines them as peptides consisting, in the mature form, of 36 or more amino acid residues with an identifiable consensus sequence: x 2 _ 1 oCx 5 - 6 (G/A)xCX 3 _ 4 Cx 9 _ 13 Cx 4 _ 7 CCx n , (where x is any amino acid; Pazgier et al. Cell. MoI. Life Sci. 63 (2006) 1294-1313).
  • INSP139 Such characteristic features of ⁇ -defensin members are conserved in INSP 139.
  • INSP 139 contains two additional conserved residues, namely a glutamic acid at position 47 and an isoleucine at position 50 of the full length INSP139.
  • INSP139 splice variant (INSP139-SV) is lacking the first 21 amino acids of the mature INSP139.
  • INSP139- SV thus lacks the two conserved cysteines Cys38 and Cys40 as well as a myristoylation site.
  • INSP139-SV display common features with INSP 139 as all the other amino acids of the full length INSPl 39-SV are identical to the full length INSP 139.
  • ⁇ -defensins have been shown to form covalently bonded homodimers in vitro and/or in vivo. Such dimerization may lead to enhanced antibacterial activities.
  • the ⁇ -defensins of this invention may form dimers or oligomers. Dimers or oligomers of this invention may display enhanced activities as disclosed herein such as antibacterial activities and/or may facilitate membrane interaction and permeabilization.
  • INSP 139 polypeptides as used herein includes polypeptides comprising the INSP 139 exon 1 polypeptide, the INSP139 exon 2 polypeptide, the INSPl 39 polypeptide, INSP139 mature polypeptide, the INSP139-SV1 polypeptide, INSP139-SV1 mature polypeptide, the INSP139 His tag polypeptide, the INSP139-SV1 His tag polypeptide, the INSP139 R21 G polypeptide, the INSP139-SV1 V21G polypeptide, the INSP139 R21G His tag polypeptide and the INSP139-SV1 V21G His tag polypeptide.
  • functions as a member of the defensin family we refer to polypeptides that comprise amino acid sequence or structural features that can be identified as conserved features within the polypeptides of the defensin family, such that the polypeptide's interaction with ligand is not substantially affected detrimentally in comparison to the function of the full length wild type polypeptide.
  • Ability to function as a defensin may be measured using the assays described by Nizet et al. (Nature 2001, 414:454-457) and Cole et al. (Proc. Natl. Acad. Sci. 2002, 99(4):1813-1818).
  • the activity of a polypeptide of the present invention, alone or as part of a fusion protein, a fragment thereof and/or an antagonist thereof can be confirmed in at least one of the following assays:
  • Each of the ⁇ -defensins characterized has the capacity to kill or inhibit in vitro a wide variety of bacteria (Gram negative or Gram positive) and fungi.
  • activity of ⁇ -defensins of this invention can be for example determined by measuring in vitro the inhibition of bacteria and fungi in the presence of INSP139.
  • the bacteria are selected from Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Candida albicans, Candida parapsilosis, Candida krusei, Enterococcus faecalis, Streptococcus pyogenes, Staphylococcus carnosus, Burkholderia cepacia and/or Saccharomyces cerevisiae.
  • Activity of ⁇ -defensins polypeptides of this invention may also be confirmed using the assay of Yadava et al. on the antimicrobial activities of human ⁇ -defensins against Bacillus species (Yadava et al. Int J Antimicrob Agents. 2006 Jun 20).
  • Antiviral activities, ⁇ -defensins can directly inactivate HIV-I virions, vaccinia viruses, adenoviruses, rhinoviruses, influenza viruses and Parainfluenza viruses. This activity appears to be a common property of defensins from different families.
  • activity of ⁇ -defensins of this invention can be for example determined by measuring the immunodeficiency virus-type 1 (HIV)-I replication in vivo in the presence of INSP 139, or by the INSP 139 dose-dependent inhibition of HIV-I replication in vitro (see Pazgier et al.), or by measuring the inhibitory effect on haemagglutinin (HA)-mediated fusion and membrane-protein mobility in pre- treatment of either HA-expressing cells or target cells (Klotman & Chang. Nat Rev Immunol. 2006 Jun;6(6):447-56), or INSP139's activity on the replication of vaccinia viruses, adenoviruses, rhinoviruses, influenza viruses and/or Parainfluenza viruses.
  • HIV immunodeficiency virus-type 1
  • HA haemagglutinin
  • ⁇ -defensins have been shown to recruit human immature dendritic cells (DCs), memory peripheral blood memory T cells and monocytes, and to activate mast cells and neutrophils.
  • DCs dendritic cells
  • ⁇ - defensin-2 has multiple activities on mast cells, including induction of mast-cell migration, degranulation and prostaglandin D 2 production.
  • the ability of ⁇ - defensins to chemoattract and/or activate a wide variety of blood cells suggests that ⁇ -defensins play roles in both the innate and antigen-specific immunity of the host (Pazgier et al).
  • ⁇ -defensins contribute to innate and adaptive immunity through the production of pro-inflammatory cytokines such as IL-8, IL-18 and IL-20, and of the cathelicidin LL-37 (Pazgier et al).
  • pro-inflammatory cytokines such as IL-8, IL-18, IL-20
  • cathelicidin LL-37 Pazgier et al.
  • IL-8, IL-18, IL-20 are believed to be involved in the pathogenesis of psoriasis, ⁇ -defensins may also activate the classical complement pathway.
  • activity of ⁇ -defensins of this invention can be determined by measuring recruitment of DCs, memory peripheral blood memory T cells and/or monocytes, and/or activation of mast cells and/or neutrophils in the presence INSP 139, for example induction of mast-cell migration, degranulation and/or prostaglandin D 2 production.
  • Activity of ⁇ -defensins of this invention may also be determined by measuring production of pro-inflammatory cytokines such as IL-8, IL-18 and/or IL-20 and/or of the cathelicidin LL-37 in the presence of INSP 139.
  • ⁇ -defensins of this invention can be determined by measuring cell differentiation processes during the maturation of keratinocytes in the presence of INSP139.
  • ⁇ -defensin-3 expression in osteoarthritic (OA) joints in the absence of bacterial challenge prompted suggestion of the role of this peptide in the tissue remodeling processes taking place in OA cartilages (Pazgier et al).
  • activity of ⁇ -defensins of this invention can be determined by measuring tissue remodeling processes taking place in OA cartilages in the presence of INSP139.
  • a rat ⁇ -defensin has been shown to enhance sperm maturation by inducing Ca 2+ uptake and subsequent motility and progressive movement of immature spermatozoa.
  • a macaque ⁇ -defensin coats the entire ejaculated sperm and masks zona pellucida ligands on the sperm surface but becomes dissociated when sperm are fully capacitated, suggesting that this beta- defensin may be an important decapacitation factor on the sperm surface that needs to be removed before sperm-zona interaction and fertilization (Patil et ah).
  • activity of ⁇ -defensins of this invention can be determined by measuring protective, adhesive, and/or regulatory functions in the reproductive tract in the presence of INSP 139.
  • Activity of ⁇ -defensins of this invention may also be determined in sperm maturation by inducing Ca 2+ uptake and subsequent motility and/or progressive movement of immature spermatozoa in the presence of INSP139.
  • a preferred assay is the radial diffusion assay for the measurement of antimicrobial activity as described in Yadava et al. (Int J Antimicrob Agents. 2006 Jun 20).
  • clearing zones are measured from diffusion of the ⁇ -defensin polypeptides of this invention through the agar medium and subsequent inhibition of bacterial growth.
  • inhibition is tested for B. anthracis, B. thuringiensis, B. licheniformis and/or B. cereus.
  • the clearing zones are measured in clearing units.
  • the ⁇ -defensin polypeptides of this invention display at least 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 100, 120 or more clearing units.
  • the incubation time is at least 0.5, 1, 2, 3, 4, 5, 8, 10 or more hours.
  • the EC50 value of the ⁇ -defensin polypeptides of this invention is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60 or more ⁇ g/mL.
  • the polypeptides of the first aspect of the invention may further comprise a histidine tag.
  • the histidine tag is found at the C-terminal of the polypeptide.
  • the histidine tag comprises 1-10 histidine residues (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues). More preferably the histidine tag comprises 6 histidine residues.
  • an “antigenic determinant” of the present invention may be a part of a polypeptide of the present invention, which binds to an antibody-combining site or to a T-cell receptor (TCR).
  • an "antigenic determinant” may be a site on the surface of a polypeptide of the present invention to which a single antibody molecule binds.
  • an antigen has several or many different antigenic determinants and reacts with antibodies of many different specificities.
  • the antibody is immunospecific to a polypeptide of the invention.
  • the antibody is immunospecific to a polypeptide of the invention, which is not part of a fusion protein.
  • the antibody is immunospecific to INSP 139 or a fragment thereof.
  • Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • the "antigenic determinant” refers to a particular chemical group on a polypeptide of the present invention that is antigenic, i.e. that elicit a specific immune response.
  • the invention provides a purified nucleic acid molecule which encodes a polypeptide of the first aspect of the invention.
  • purified nucleic acid molecule preferably refers to a nucleic acid molecule of the invention that (1) has been separated from at least about 50 percent of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells, (2) is not linked to all or a portion of a polynucleotide to which the "purified nucleic acid molecule" is linked in nature, (3) is operably linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature as part of a larger polynucleotide sequence.
  • the isolated nucleic acid molecule of the present invention is substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use.
  • genomic DNA are specifically excluded from the scope of the invention.
  • genomic DNA larger than 10 kbp (kilo base pairs), 50 kbp, 100 kbp, 150 kbp, 200 kbp, 250 kbp or 300 kbp are specifically excluded from the scope of the invention.
  • the "purified nucleic acid molecule" consists of cDNA only.
  • the purified nucleic acid molecule comprises the nucleic acid sequence as recited in SEQ ID NO:1 (encoding the INSP139 exon 1 polypeptide), SEQ ID NO:3 (encoding the INSP139 exon 2 polypeptide), SEQ ID NO:5 (encoding the INSP139 polypeptide), SEQ ID NO:7 (encoding the INSP139 mature polypeptide), SEQ ID NO: 9 (encoding the INSP139-SV1 polypeptide) and/or SEQ ID NO: 11 (encoding the INSP 139- SVl mature polypeptide) or is a redundant equivalent or fragment of any one of these sequences.
  • the invention further provides that the purified nucleic acid molecule consists of the nucleic acid sequence as recited in SEQ ID NO:1 (encoding the INSP 139 exon 1 polypeptide), SEQ ID NO:3 (encoding the INSP139 exon 2 polypeptide), SEQ ID NO:5 (encoding the INSP139 polypeptide), SEQ ID NO:7 (encoding the INSP139 mature polypeptide), SEQ ID NO: 9 (encoding the INSP139-SV1 polypeptide) and/or SEQ ID NO: 11 (encoding the INSP139-SV1 mature polypeptide) or is a redundant equivalent or fragment of any one of these sequences.
  • the invention provides a purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of the second aspect of the invention.
  • High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (15OmM NaCl, 15mM trisodium citrate), 5OmM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C.
  • the invention provides a vector, such as an expression vector, that contains a nucleic acid molecule of the second or third aspect of the invention.
  • the invention provides a host cell transformed with a vector of the fourth aspect of the invention.
  • the invention provides a ligand which binds specifically to protein members of the defensin family of the first aspect of the invention.
  • the ligand inhibits the function of a polypeptide of the first aspect of the invention which is a member of the defensin family of proteins.
  • Ligands to a polypeptide according to the invention may come in various forms, including natural or modified substrates, enzymes, receptors, small organic molecules such as small natural or synthetic organic molecules of up to 2000Da, preferably 800Da or less, peptidomimetics, inorganic molecules, peptides, polypeptides, antibodies, structural or functional mimetics of the aforementioned.
  • the invention provides a compound that is effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
  • Such compounds may be identified using the assays and screening methods disclosed herein.
  • a compound of the seventh aspect of the invention may either increase (agonise) or decrease (antagonise) the level of expression of the gene or the activity of the polypeptide.
  • the identification of the functions of the INSPl 39 polypeptides allows for the design of screening methods capable of identifying compounds that are effective in the treatment and/or diagnosis of disease.
  • Ligands and compounds according to the sixth and seventh aspects of the invention may be identified using such methods. These methods are included as aspects of the present invention.
  • Another aspect of this invention resides in the use of an INSP 139 gene or polypeptide as a target for the screening of candidate drug modulators, particularly candidate drugs active against defensin related disorders.
  • a further aspect of this invention resides in methods of screening of compounds for therapy of defensin related disorders, comprising determining the ability of a compound to bind to an INSP 139 gene or polypeptide, or a fragment thereof.
  • a further aspect of this invention resides in methods of screening of compounds for therapy of defensin related disorders, comprising testing for modulation of the activity of an INSP 139 gene or polypeptide, or a fragment thereof.
  • the invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in therapy or diagnosis of diseases in which members of the defensin family are implicated.
  • Such diseases may include cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis' sarcoma; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellitus, osteoporosis, and obesity, AIDS and renal disease; infections including viral infection, bacterial infection, fungal infection and parasitic infection
  • the disease is one in which the defensin family of proteins is implicated, such as sublethal endotoxaemia, septic shock, microbial infection of the amniotic cavity, Jarish-Herxheimer reaction of relapsing fever, infectious diseases of the central nervous system, acute pancreatitis, ulcerative colitis, empyaema, haemolytic uraemic syndrome, meningococcal disease, gastric infection, pertussis, peritonitis, psoriasis, rheumatoid arthritis, sepsis, asthma, HIV, AIDS, glomerulonephritis, otitis media, paranasal sinusitis, labyrinthitis, meningitis, reproductive health disorders, infertility or male infertility.
  • the defensin family of proteins is implicated, such as sublethal endotoxaemia, septic shock, microbial infection of the amniotic cavity, Jarish-
  • the disease is one caused by infection with bacteria selected from Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus Aureus, Streptococcus pneumoniae, Candida albicans, Candida parapsilosis, Candida krusei, Enterococcus faecalis, Streptococcus pyogenes, Staphylococcus carnosus, Bacillus species such as B. anthracis, B. thuringiensis, B. licheniformis or B.
  • a virus selected from HIV, vaccinia virus, adenovirus, rhinovirus, influenza virus and/or Parainfluenza virus.
  • the moieties of the present invention may have particular utility in the therapy or diagnosis of disorders/diseases (the two terms are used interchangeably herein) of the immune system (e.g. an autoimmune disease), malignancies, inflammatory disorders, asthma and rheumatoid arthritis.
  • disorders/diseases the two terms are used interchangeably herein
  • the immune system e.g. an autoimmune disease
  • malignancies e.g. an autoimmune disease
  • inflammatory disorders e.g. asthma and rheumatoid arthritis.
  • the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide of the first aspect of the invention or the activity of a polypeptide of the first aspect of the invention in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.
  • a method will preferably be carried out in vitro.
  • Similar methods may be used for monitoring the therapeutic treatment of disease in a patient, wherein altering the level of expression or activity of a polypeptide or nucleic acid molecule over the period of time towards a control level is indicative of regression of disease.
  • a preferred method for detecting polypeptides of the first aspect of the invention comprises the steps of: (a) contacting a ligand, such as an antibody, of the sixth aspect of the invention with a biological sample under conditions suitable for the formation of a ligand- polypeptide complex; and (b) detecting said complex.
  • a number of different such methods according to the ninth aspect of the invention exist, as the skilled reader will be aware, such as methods of nucleic acid hybridization with short probes, point mutation analysis, polymerase chain reaction (PCR) amplification and methods using antibodies to detect aberrant protein levels. Similar methods may be used on a short or long term basis to allow therapeutic treatment of a disease to be monitored in a patient.
  • the invention also provides kits that are useful in these methods for diagnosing disease.
  • the invention provides for the use of a polypeptide of the first aspect of the invention as a defensin protein.
  • Suitable uses of the polypeptides of the invention as defensin proteins include use as a regulator of cellular growth, for example, against Gram- positive and Gram-negative bacteria, mycobacteria, fungi and even enveloped viruses (In vitro defensins have been shown to be active against E. coli, Listeria monocytogenes, Salmonella typhimurium and Candida albicans). High concentrations may be cytotoxic for mammalian cells. Lower concentrations may promote growth in epithelial cells and fibroblasts, thus suggesting a role in wound healing.
  • Defensins have also been shown to be chemotactic for monocytes, polymorphonuclear leucocytes and T-cells and may play a role in adaptive immunity (Yang, D. et al. 2002, Trends Immunol. 23(6):291-6).
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, in conjunction with a pharmaceutically- acceptable carrier.
  • the present invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease, including, but not limited to, sublethal endotoxaemia, septic shock, microbial infection of the amniotic cavity, Jarish-Herxheimer reaction of relapsing fever, infectious diseases of the central nervous system, acute pancreatitis, ulcerative colitis, empyaema, haemolytic uraemic syndrome, meningococcal disease, gastric infection, pertussis, peritonitis, psoriasis, rheumatoid arthritis, sepsis, asthma, HIV,
  • the invention provides a method of treating a disease in a patient comprising administering to the patient a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention.
  • the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an agonist.
  • the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an antagonist.
  • antagonists include antisense nucleic acid molecules, ribozymes and ligands, such as antibodies.
  • the INSP 139 polypeptides are members of the defensin family of proteins and thus have roles in many disease states. Antagonists of the INSPl 39 polypeptides are of particular interest as they provide a way of modulating these disease states.
  • the invention provides transgenic or knockout non-human animals that have been transformed to express higher, lower or absent levels of a polypeptide of the first aspect of the invention.
  • Such transgenic animals are very useful models for the study of disease and may also be used in screening regimes for the identification of compounds that are effective in the treatment or diagnosis of such a disease.
  • “functional equivalent” refers to a protein or nucleic acid molecule that possesses functional or structural characteristics that are substantially similar to a polypeptide or nucleic acid molecule of the present invention.
  • a functional equivalent of a protein may contain modifications depending on the necessity of such modifications for the performance of a specific function.
  • the term “functional equivalent” is intended to include the fragments, mutants, hybrids, variants, analogs, or chemical derivatives of a molecule.
  • the "functional equivalent” may be a protein or nucleic acid molecule that exhibits any one or more of the functional activities of the polypeptides of the present invention.
  • the "functional equivalent” may be a protein or nucleic acid molecule that displays substantially similar activity compared with INSP 139 or fragments thereof in a suitable assay for the measurement of biological activity or function.
  • the "functional equivalent” may be a protein or nucleic acid molecule that displays identical or higher activity compared with INSP 139 or fragments thereof in a suitable assay for the measurement of biological activity or function.
  • the “functional equivalent” may be a protein or nucleic acid molecule that displays 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100% or more activity compared with INSP 139 or fragments thereof in a suitable assay for the measurement of biological activity or function.
  • the "functional equivalent” may be a protein or polypeptide capable of exhibiting a substantially similar in vivo or in vitro activity as the polypeptides of the invention.
  • the "functional equivalent” may be a protein or polypeptide capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion of the polypeptides of the invention would.
  • a "functional equivalent” would be able, in an immunoassay, to diminish the binding of an antibody to the corresponding peptide ⁇ i.e., the peptide the amino acid sequence of which was modified to achieve the "functional equivalent") of the polypeptide of the invention, or to the polypeptide of the invention itself, where the antibody was raised against the corresponding peptide of the polypeptide of the invention.
  • An equimolar concentration of the functional equivalent will diminish the aforesaid binding of the corresponding peptide by at least about 5%, preferably between about 5% and 10%, more preferably between about 10% and 25%, even more preferably between about 25% and 50%, and most preferably between about 40% and 50%.
  • polypeptide includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. This term refers both to short chains (peptides and oligopeptides) and to longer chains (proteins).
  • the polypeptide of the present invention may be in the form of a mature protein or may be a pre-, pro- or prepro- protein that can be activated by cleavage of the pre-, pro- or prepro- portion to produce an active mature polypeptide.
  • the pre-, pro- or prepro- sequence may be a leader or secretory sequence or may be a sequence that is employed for purification of the mature polypeptide sequence.
  • the polypeptide of the first aspect of the invention may form part of a fusion protein.
  • a fusion protein may contain one or more additional amino acid sequences which may contain secretory or leader sequences, pro-sequences, sequences which aid in purification, or sequences that confer higher protein stability, for example during recombinant production.
  • the mature polypeptide may be fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol).
  • a polypeptide of the invention that may comprise a sequence having at least 85% of homology with INSP139, is a fusion protein.
  • fusion proteins can be obtained by cloning a polynucleotide encoding a polypeptide comprising a sequence having at least 85% of homology with INSP 139 in frame to the coding sequences for a heterologous protein sequence.
  • heterologous when used herein, is intended to designate any polypeptide other than a human INSP 139 polypeptide.
  • heterologous sequences that can be comprised in the fusion proteins either at the N- or C-terminus, include: extracellular domains of membrane-bound protein, immunoglobulin constant regions (Fc regions), multimerization domains, domains of extracellular proteins, signal sequences, export sequences, and sequences allowing purification by affinity chromatography.
  • heterologous sequences are commercially available in expression plasmids since these sequences are commonly included in fusion proteins in order to provide additional properties without significantly impairing the specific biological activity of the protein fused to them (Terpe K, 2003, Appl Microbiol Biotechnol, 60:523-33).
  • heterologous sequence can be eliminated by a proteolytic cleavage, for example by inserting a proteolytic cleavage site between the protein and the heterologous sequence, and exposing the purified fusion protein to the appropriate protease.
  • the INSP 139 polypeptide may be purified by means of a hexa-histidine peptide fused at the C-terminus of INSP 139.
  • the fusion protein comprises an immunoglobulin region
  • the fusion may be direct, or via a short linker peptide which can be as short as 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues in length.
  • Said linker maybe a tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a 13 -amino acid linker sequence comprising GIu- Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met (SEQ ID NO: 19) introduced between the sequence of the substances of the invention and the immunoglobulin sequence.
  • the resulting fusion protein has improved properties, such as an extended residence time in body fluids (i.e. an increased half-life), increased specific activity, increased expression level, or the purification of the fusion protein is facilitated.
  • the protein is fused to the constant region of an Ig molecule.
  • it is fused to heavy chain regions, like the CH2 and CH3 domains of human IgGl, for example.
  • Other isoforms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as isoforms IgG2 or IgG4, or other Ig classes, like IgM or IgA, for example. Fusion proteins may be monomelic or multimeric, hetero- or homomultimeric.
  • the functional derivative comprises at least one moiety attached to one or more functional groups, which occur as one or more side chains on the amino acid residues.
  • the moiety is a polyethylene (PEG) moiety. PEGylation may be carried out by known methods, such as the ones described in WO99/55377, for example.
  • Polypeptides may contain amino acids other than the 20 gene-encoded amino acids, modified either by natural processes, such as by post-translational processing or by chemical modification techniques which are well known in the art.
  • modifications which may commonly be present in polypeptides of the present invention are glycosylation, lipid attachment, sulphation, gamma-carboxylation, for instance of glutamic acid residues, hydroxylation and ADP-ribosylation.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini, hi fact, blockage of the amino or carboxyl terminus in a polypeptide, or both, by a covalent modification is common in naturally-occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention.
  • modifications that occur in a polypeptide often will be a function of how the polypeptide is made.
  • the nature and extent of the modifications in large part will be determined by the post-translational modification capacity of the particular host cell and the modification signals that are present in the amino acid sequence of the polypeptide in question. For instance, glycosylation patterns vary between different types of host cell.
  • polypeptides of the present invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally-occurring polypeptides (for example purified from cell culture), recombinantly-produced polypeptides (including fusion proteins), synthetically-produced polypeptides or polypeptides that are produced by a combination of these methods.
  • the functionally-equivalent polypeptides of the first aspect of the invention may be polypeptides that are homologous to the INSP139 polypeptides. Two polypeptides are said to be "homologous", as the term is used herein, if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide.
  • Homologous polypeptides therefore include natural biological variants (for example, allelic variants or geographical variations within the species from which the polypeptides are derived) and mutants (such as mutants containing amino acid substitutions, insertions or deletions) of the INSP 139 polypeptides.
  • Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code.
  • Such substitutions are among Ala, VaI, Leu and He; among Ser and Thr; among the acidic residues Asp and GIu; among Asn and GIn; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr.
  • Particularly preferred are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination.
  • silent substitutions, additions and deletions which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions.
  • Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group.
  • any substitution should be preferably a "conservative” or “safe” substitution, which is commonly defined a substitution introducing an amino acids having sufficiently similar chemical properties ⁇ e.g. a basic, positively charged amino acid should be replaced by another basic, positively charged amino acid), in order to preserve the structure and the biological function of the molecule.
  • non-conservative mutations can be also introduced in the polypeptides of the invention with different purposes. Mutations reducing the affinity of the defensin protein may increase its ability to be reused and recycled, potentially increasing its therapeutic potency (Robinson CR, 2002). Immunogenic epitopes eventually present in the polypeptides of the invention can be exploited for developing vaccines (Stevanovic S, 2002), or eliminated by modifying their sequence following known methods for selecting mutations for increasing protein stability, and correcting them (van den Burg B and Eijsink V 5 2002; WO 02/05146, WO 00/34317, WO 98/52976).
  • amino acids derivatives included in peptide mimetics are those defined in Table 2.
  • a non-exhaustive list of amino acid derivatives also include aminoisobutyric acid (Aib), hydroxyproline (Hyp), 1,2,3,4-tetrahydro-isoquinoline- 3-COOH, indoline-2carboxylic acid, 4-difluoro-proline, L- thiazolidme-4-carboxylic acid, L-homoproline, 3,4-dehydro-proline, 3,4-dihydroxy-phenylalanine, cyclohexyl-glycine, and phenylglycine.
  • amino acid derivative is intended an amino acid or amino acid-like chemical entity other than one of the 20 genetically encoded naturally occurring amino acids, hi particular, the amino acid derivative may contain substituted or non-substituted, linear, branched, or cyclic alkyl moieties, and may include one or more heteroatoms.
  • the amino acid derivatives can be made de novo or obtained from commercial sources (Calbiochem- Novabiochem AG, Switzerland; Bachem, USA).
  • polypeptides of the first aspect of the invention have a degree of sequence identity with the INSP 139 polypeptides, or with active fragments thereof, of greater than 80%. More preferred polypeptides have degrees of identity of greater than 85%, 90%, 95%, 98% or 99%, respectively.
  • the functionally-equivalent polypeptides of the first aspect of the invention may also be polypeptides which have been identified using one or more techniques of structural alignment.
  • the Inpharmatica Genome Threader technology that forms one aspect of the search tools used to generate the BiopendiumTM search database may be used (see PCT application WO 01/69507) to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the INSP 139 polypeptides, are predicted to be members of the defensin family, by virtue of sharing significant structural homology with the INSP 139 polypeptide sequences.
  • significant structural homology is meant that the Inpharmatica Genome Threader predicts two proteins to share structural homology with a certainty of 10% and above.
  • polypeptides of the first aspect of the invention also include fragments of the INSP 139 polypeptides and fragments of the functional equivalents of the INSP 139 polypeptides, provided that those fragments are members of the defensin family or have an antigenic determinant in common with the INSP139 polypeptides.
  • fragment refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the INSP 139 polypeptides or one of their functional equivalents.
  • the fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). Small fragments may form an antigenic determinant.
  • Nucleic acid fragments according to the invention are preferably 10-250 nucleotides in length, preferably 20-200 nucleotides, preferably 50-150 nucleotides, preferably 75-100 nucleotides in length.
  • Polypeptide fragments according to the invention are preferably 5-80 amino acids in length, preferably 10-70, preferably 25-60, preferably 40-50 amino acids in length.
  • amino acid fragments according to the invention are 50, 45, 40 or 39 amino acids in length, or smaller.
  • Fragments of the full length INSP 139 polypeptides may consist of combinations of 1 or 2 of neighbouring exon sequences in the INSP 139 polypeptide sequences, respectively. For example, such combinations include exons 1 and 2 of the INSP 139 polypeptide. Such fragments are included in the present invention. Such fragments may also comprise a ⁇ - defensin domain such as the domains spanning amino acids residues 23 to 64, 21 to 61 or 31 to 60 ofINSP139.
  • Preferred active ⁇ -defensin peptides or fragments of INSP 139 span residues 23 to 64, residues 27 to 61, residues 29 to 66, residues 29 to 62 or residues 31 to 60 of full length INSP139 (SEQ ID NO: 6).
  • Preferred active ⁇ -defensin peptides or fragments of INSP139-SV span residues 20 to 45, residues 20 to 43, residues 20 to 41, residues 20 to 40, or residues 20 to 39 of full length INSPl 39-SV (SEQ ID NO: 10).
  • fragments may be "free-standing", i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region.
  • the fragment of the invention When comprised within a larger polypeptide, the fragment of the invention most preferably forms a single continuous region.
  • certain preferred embodiments relate to a fragment having a pre- and/or pro- polypeptide region fused to the amino terminus of the fragment and/or an additional region fused to the carboxyl terminus of the fragment.
  • several fragments may be comprised within a single larger polypeptide.
  • polypeptides of the present invention or their immunogenic fragments can be used to generate ligands, such as polyclonal or monoclonal antibodies, that are immunospecif ⁇ c for the polypeptides.
  • ligands such as polyclonal or monoclonal antibodies
  • Such antibodies may be employed to isolate or to identify clones expressing the polypeptides of the invention or to purify the polypeptides by affinity chromatography.
  • the antibodies may also be employed as diagnostic or therapeutic aids, amongst other applications, as will be apparent to the skilled reader.
  • immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
  • antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')2 and Fv, which are capable of binding to the antigenic determinant in question. Such antibodies thus bind to the polypeptides of the first aspect of the invention.
  • substantially greater affinity we mean that there is a measurable increase in the affinity for a polypeptide of the invention as compared with the affinity for known secreted proteins.
  • the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 10 3 -fold, 10 4 - fold, 10 5 -fold, 10 6 -fold or greater for a polypeptide of the invention than for known secreted proteins such as members of the defensin family of proteins.
  • a selected mammal such as a mouse, rabbit, goat or horse
  • a polypeptide of the first aspect of the invention may be immunised with a polypeptide of the first aspect of the invention.
  • the polypeptide used to immunise the animal can be derived by recombinant DNA technology or can be synthesized chemically.
  • the polypeptide can be conjugated to a carrier protein.
  • Commonly used carriers to which the polypeptides may be chemically coupled include bovine serum albumin, thyroglobulin and keyhole limpet haemocyanin.
  • the coupled polypeptide is then used to immunise the animal. Serum from the immunised animal is collected and treated according to known procedures, for example by immunoaffinity chromatography.
  • Monoclonal antibodies to the polypeptides of the first aspect of the invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies using hybridoma technology is well known (see, for example, Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, 11-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985).
  • Panels of monoclonal antibodies produced against the polypeptides of the first aspect of the invention can be screened for various properties, i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies are particularly useful in purification of the individual polypeptides against which they are directed. Alternatively, genes encoding the monoclonal antibodies of interest may be isolated from hybridomas, for instance by PCR techniques known in the art, and cloned and expressed in appropriate vectors.
  • Chimeric antibodies in which non-human variable regions are joined or fused to human constant regions (see, for example, Liu et al, Proc. Natl. Acad. Sci. USA, 84, 3439 (1987)), may also be of use.
  • the antibody may be modified to make it less immunogenic in an individual, for example by humanisation (see Jones et al., Nature, 321, 522 (1986); Verhoeyen et al., Science, 239, 1534 (1988); Kabat et al, J. Immunol., 147, 1709 (1991); Queen et al, Proc. Natl Acad. Sci. USA, 86, 10029 (1989); Gorman et al, Proc. Natl Acad. Sci. USA, 88, 34181 (1991); and Hodgson et al, Bio/Technology, 9, 421 (1991)).
  • humanised antibody refers to antibody molecules in which the CDR amino acids and selected other amino acids in the variable domains of the heavy and/or light chains of a non-human donor antibody have been substituted in place of the equivalent amino acids in a human antibody.
  • the humanised antibody thus closely resembles a human antibody but has the binding ability of the donor antibody.
  • the antibody may be a "bispecific" antibody, that is an antibody having two different antigen binding domains, each domain being directed against a different epitope.
  • Phage display technology may be utilised to select genes which encode antibodies with binding activities towards the polypeptides of the invention either from repertoires of PCR amplified V-genes of lymphocytes from humans screened for possessing the relevant antibodies, or from naive libraries (McCafferty, J. et al, (1990), Nature 348, 552-554; Marks, J. et al, (1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al, (1991) Nature 352, 624-628).
  • Antibodies generated by the above techniques have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).
  • the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme.
  • nucleic acid molecules of the second and third aspects of the invention are those which encode a polypeptide sequence as recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ E) NO: 16, SEQ E) NO: 17 and SEQ E) NO: 18 and functionally equivalent polypeptides.
  • These nucleic acid molecules may be used in the methods and applications described herein.
  • the nucleic acid molecules of the invention preferably comprise at least n consecutive nucleotides from the sequences disclosed herein where, depending on the particular sequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
  • nucleic acid molecules of the invention also include sequences that are complementary to nucleic acid molecules described above (for example, for antisense or probing purposes).
  • Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance cDNA, synthetic DNA or genomic DNA. Such nucleic acid molecules may be obtained by cloning, by chemical synthetic techniques or by a combination thereof. The nucleic acid molecules can be prepared, for example, by chemical synthesis using techniques such as solid phase phosphoramidite chemical synthesis, from genomic or cDNA libraries or by separation from an organism. RNA molecules may generally be generated by the in vitro or in vivo transcription of DNA sequences.
  • the nucleic acid molecules may be double-stranded or single-stranded.
  • Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the anti-sense strand.
  • nucleic acid molecule also includes analogues of DNA and RNA, such as those containing modified backbones, and peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63).
  • a nucleic acid molecule which encodes a polypeptide of this invention may be identical to the coding sequence of one or more of the nucleic acid molecules disclosed herein.
  • These molecules also may have a different sequence which, as a result of the degeneracy of the genetic code, encodes a polypeptide as recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10 SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.
  • nucleic acid molecules may include, but are not limited to, the coding sequence for the mature polypeptide by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pro-, pre- or prepro- polypeptide sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with further additional, non-coding sequences, including non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription (including termination signals), ribosome binding and mRNA stability.
  • the nucleic acid molecules may also include additional sequences which encode additional amino acids, such as those which provide additional functionalities.
  • nucleic acid molecules of the second and third aspects of the invention may also encode the fragments or the functional equivalents of the polypeptides and fragments of the first aspect of the invention.
  • a nucleic acid molecule may be a naturally-occurring variant such as a naturally-occurring allelic variant, or the molecule may be a variant that is not known to occur naturally.
  • non-naturally occurring variants of the nucleic acid molecule may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.
  • variants in this regard are variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or insertions.
  • the substitutions, deletions or insertions may involve one or more nucleotides.
  • the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or insertions.
  • the nucleic acid molecules of the invention can also be engineered, using methods generally known in the art, for a variety of reasons, including modifying the cloning, processing, and/or expression of the gene product (the polypeptide).
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides are included as techniques which may be used to engineer the nucleotide sequences.
  • Site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations and so forth.
  • Nucleic acid molecules which encode a polypeptide of the first aspect of the invention may be ligated to a heterologous sequence so that the combined nucleic acid molecule encodes a fusion protein.
  • Such combined nucleic acid molecules are included within the second or third aspects of the invention.
  • a fusion protein that can be recognised by a commercially-available antibody.
  • a fusion protein may also be engineered to contain a cleavage site located between the sequence of the polypeptide of the invention and the sequence of a heterologous protein so that the polypeptide may be cleaved and purified away from the heterologous protein.
  • the nucleic acid molecules of the invention also include antisense molecules that are partially complementary to nucleic acid molecules encoding polypeptides of the present invention and that therefore hybridize to the encoding nucleic acid molecules (hybridization).
  • antisense molecules such as oligonucleotides, can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide of the invention, as will be known by those of ordinary skill in the art (see, for example, Cohen, J.S., Trends in Pharm. Sci., 10, 435 (1989), Okano, J. Neurochem. 56, 560 (1991); O'Connor, J. Neurochem 56, 560 (1991); Lee et al, Nucleic Acids Res 6, 3073 (1979); Cooney et al, Science 241, 456 (1988); Dervan et al, Science 251, 1360 (1991).
  • hybridization refers to the association of two nucleic acid molecules with one another by hydrogen bonding. Typically, one molecule will be fixed to a solid support and the other will be free in solution. Then, the two molecules may be placed in contact with one another under conditions that favour hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase molecule to the solid support (Denhardt's reagent or BLOTTO); the concentration of the molecules; use of compounds to increase the rate of association of molecules (dextran sulphate or polyethylene glycol); and the stringency of the washing conditions following hybridization (see Sambrook et al. [supra]).
  • the inhibition of hybridization of a completely complementary molecule to a target molecule may be examined using a hybridization assay, as known in the art (see, for example, Sambrook et al. [supra]).
  • a substantially homologous molecule will then compete for and inhibit the binding of a completely homologous molecule to the target molecule under various conditions of stringency, as taught in WaM, G.M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A.R. (1987; Methods Enzymol. 152:507-511).
  • Stringency refers to conditions in a hybridization reaction that favour the association of very similar molecules over association of molecules that differ.
  • High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (15OmM NaCl, 15mM trisodium citrate), 5OmM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C.
  • Low stringency conditions involve the hybridisation reaction being carried out at 35°C (see Sambrook et al. [supra]).
  • the conditions used for hybridization are those of high stringency.
  • nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding the INSP 139 polypeptides and nucleic acid molecules that are substantially complementary to such nucleic acid molecules.
  • a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to such coding sequences, or is a nucleic acid molecule that is complementary thereto.
  • nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98%, 99% or more identical over their entire length to the same are particularly preferred.
  • Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the INSP 139 polypeptides.
  • the invention also provides a process for detecting a nucleic acid molecule of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting any such duplexes that are formed.
  • a nucleic acid molecule as described above may be used as a hybridization probe for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs and genomic clones encoding the INSP 139 polypeptides and to isolate cDNA and genomic clones of homologous or orthologous genes that have a high sequence similarity to the gene encoding this polypeptide.
  • the sequencing process may be automated using machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • One method for isolating a nucleic acid molecule encoding a polypeptide with an equivalent function to that of the INSP 139 polypeptide is to probe a genomic or cDNA library with a natural or artificially-designed probe using standard procedures that are recognised in the art (see, for example, "Current Protocols in Molecular Biology", Ausubel et al. (eds). Greene Publishing Association and John Wiley Interscience, New York, 1989,1992).
  • Probes comprising at least 15, preferably at least 30, and more preferably at least 50, contiguous bases that correspond to, or are complementary to, nucleic acid sequences from the appropriate encoding gene (SEQ ID NO:1, SEQ ID NO:3, SEQ JD NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO: 11), are particularly useful probes. Such probes may be labelled with an analytically-detectable reagent to facilitate their identification.
  • Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes and enzymes that are capable of catalysing the formation of a detectable product.
  • probes the ordinarily skilled artisan will be capable of isolating complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding proteins of interest from human, mammalian or other animal sources and screening such sources for related sequences, for example, for additional members of the family, type and/or subtype.
  • isolated cDNA sequences will be incomplete, in that the region encoding the polypeptide will be cut short, normally at the 5' end.
  • Several methods are available to obtain full length cDNAs, or to extend short cDNAs. Such sequences may be extended utilising a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed is based on the method of Rapid Amplification of cDNA Ends (RACE; see, for example, Frohman et al, PNAS USA 85, 8998-9002, 1988).
  • RACE Rapid Amplification of cDNA Ends
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic, 1, 111-119). Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991); Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • libraries that have been size- selected to include larger cDNAs.
  • random-primed libraries are preferable, in that they will contain more sequences that contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • the nucleic acid molecules of the present invention may be used for chromosome localisation.
  • a nucleic acid molecule is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important step in the confirmatory correlation of those sequences with the gene-associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • the relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localised by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleic acid molecule may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
  • the nucleic acid molecules of the present invention are also valuable for tissue localisation.
  • Such techniques allow the determination of expression patterns of the polypeptide in tissues by detection of the mRNAs that encode them.
  • These techniques include in situ hybridization techniques and nucleotide amplification techniques, such as PCR. Results from these studies provide an indication of the normal functions of the polypeptide in the organism.
  • comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by a mutant gene provide valuable insights into the role of mutant polypeptides in disease. Such inappropriate expression may be of a temporal, spatial or quantitative nature.
  • RNA interference (Elbashir, SM et al, Nature 2001, 411, 494-498) is one method of sequence specific post- transcriptional gene silencing that may be employed. Short dsRNA oligonucleotides are synthesised in vitro and introduced into a cell. The sequence specific binding of these dsRNA oligonucleotides triggers the degradation of target niRNA, reducing or ablating target protein expression.
  • Efficacy of the gene silencing approaches assessed above may be assessed through the measurement of polypeptide expression (for example, by Western blotting), and at the RNA level using TaqMan-based methodologies.
  • the vectors of the present invention comprise nucleic acid molecules of the invention and may be cloning or expression vectors.
  • the host cells of the invention which may be transformed, transfected or transduced with the vectors of the invention may be prokaryotic or eukaryotic.
  • polypeptides of the invention may be prepared in recombinant form by expression of their encoding nucleic acid molecules in vectors contained within a host cell. Such expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al. ⁇ supra) and Fernandez & Hoeffler (1998, eds. "Gene expression systems. Using nature for the art of expression”. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).
  • any system or vector that is suitable to maintain, propagate or express nucleic acid molecules to produce a polypeptide in the required host may be used.
  • the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well- known and routine techniques, such as, for example, those described in Sambrook et al., ⁇ supra).
  • the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell.
  • suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids.
  • HACs Human artificial chromosomes
  • Particularly suitable expression systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (for example, baculovirus); plant cell systems transformed with virus expression vectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (for example, Ti or pBR322 plasmids); or animal cell systems.
  • Cell-free translation systems can also be employed to produce the polypeptides of the invention.
  • nucleic acid molecules encoding a polypeptide of the present invention into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al, Basic Methods in Molecular Biology (1986) and Sambrook et al, ⁇ supra). Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et al, 1989 [supra]; Ausubel et al., 1991 [supra]; Spector, Goldman & Leinwald, 1998). In eukaryotic cells, expression systems may either be transient (for example, episomal) or permanent (chromosomal integration) according to the needs of the system.
  • the encoding nucleic acid molecule may or may not include a sequence encoding a control sequence, such as a signal peptide or leader sequence, as desired, for example, for secretion of the translated polypeptide into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment.
  • a control sequence such as a signal peptide or leader sequence
  • These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • Leader sequences can be removed by the bacterial host in post-translational processing.
  • regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell.
  • regulatory sequences are those which cause the expression of a gene to be increased or decreased in response to a chemical or physical stimulus, including the presence of a regulatory compound or to various temperature or metabolic conditions.
  • Regulatory sequences are those non-translated regions of the vector, such as enhancers, promoters and 5' and 3' untranslated regions. These interact with host cellular proteins to carry out transcription and translation. Such regulatory sequences may vary in their strength and specificity. Depending on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used.
  • inducible promoters such as the hybrid lacZ promoter of the Bluescript phagemid (Stratagene, LaJoIIa, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (for example, heat shock, RUBISCO and storage protein genes) or from plant viruses (for example, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector.
  • the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
  • cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
  • Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.
  • ATCC American Type Culture Collection
  • the materials for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac” kit). These techniques are generally known to those skilled in the art and are described fully in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells.
  • all plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be utilised, so that whole plants are recovered which contain the transferred gene.
  • Practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugar cane, sugar beet, cotton, fruit and other trees, legumes and vegetables.
  • Examples of particularly preferred bacterial host cells include streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells.
  • yeast cells for example, S. cerevisiae
  • Aspergillus cells Any number of selection systems are known in the art that may be used to recover transformed cell lines. Examples include the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes that can be employed in tk " or aprt 1 * 1 cells, respectively.
  • antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dihydrofolate reductase (DHFR) that confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. MoI. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes have been described, examples of which will be clear to those of skill in the art.
  • marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed.
  • a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Expression ' of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain a nucleic acid sequence encoding a polypeptide of the invention and which express said polypeptide may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA- DNA or DNA-RNA hybridizations and protein bioassays, for example, fluorescence activated cell sorting (FACS) or immunoassay techniques (such as the enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]), that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein (see Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, MN) and Maddox, D.E. et al. (1983) J. Exp. Med, 158, 1211-1216).
  • FACS fluorescence activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • Means for producing labelled hybridization or PCR probes for detecting sequences related to nucleic acid molecules encoding polypeptides of the present invention include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled polynucleotide.
  • sequences encoding the polypeptide of the invention may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3 or SP6 and labelled nucleotides. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, MI); Promega (Madison WI); and U.S. Biochemical Corp., Cleveland, OH)).
  • Suitable reporter molecules or labels include radionuclides, enzymes and fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Nucleic acid molecules according to the present invention may also be used to create transgenic animals, particularly rodent animals. Such transgenic animals form a further aspect of the present invention. This may be done locally by modification of somatic cells, or by germ line therapy to incorporate heritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for drug molecules effective as modulators of the polypeptides of the present invention.
  • the polypeptide can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography is particularly useful for purification. Well known techniques for refolding proteins may be employed to regenerate an active conformation when the polypeptide is denatured during isolation and or purification.
  • Specialised vector constructions may also be used to facilitate purification of proteins, as desired, by joining sequences encoding the polypeptides of the invention to a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins.
  • purification-facilitating domains include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the polypeptide of the invention may be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing the polypeptide of the invention fused to several histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilised metal ion affinity chromatography as described in Porath, J. et al. (1992), Prot. Exp. Purif.
  • the polypeptide is to be expressed for use in screening assays, generally it is preferred that it be produced at the surface of the host cell in which it is expressed, hi this event, the host cells may be harvested prior to use in the screening assay, for example using techniques such as fluorescence activated cell sorting (FACS) or immunoaffmity techniques. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the expressed polypeptide. If polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • FACS fluorescence activated cell sorting
  • the present invention also provides novel targets and methods for the screening of drug candidates or leads.
  • These screening methods include binding assays and/or functional assays, and may be performed in vitro, in cell systems or in animals.
  • a particular object of this invention resides in the use of an INSP 139 polypeptide as a target for screening candidate drugs for treating or preventing defensin related disorders.
  • Another object of this invention resides in methods of selecting biologically active compounds, said methods comprising contacting a candidate compound with a INSP 139 gene or polypeptide, and selecting compounds that bind said gene or polypeptide.
  • a further other object of this invention resides in methods of selecting biologically active compounds, said method comprising contacting a candidate compound with recombinant host cell expressing a INSP 139 polypeptide with a candidate compound, and selecting compounds that bind said INSP 139 polypeptide at the surface of said cells and/or that modulate the activity of the INSP 139 polypeptide.
  • a “biologically active” compound denotes any compound having biological activity in a subject, preferably therapeutic activity, more preferably a compound having defensin activity, and further preferably a compound that can be used for treating INSP 139 related disorders, or as a lead to develop drugs for treating a defensin related disorder.
  • a "biologically active” compound preferably is a compound that modulates the activity of INSP139.
  • the above methods may be conducted in vitro, using various devices and conditions, including with immobilized reagents, and may further comprise an additional step of assaying the activity of the selected compounds in a model of defensin related disorders, such as an animal model.
  • Preferred selected compounds are agonists of INSP 139, i.e., compounds that can bind to INSP 139 and mimic the activity of an endogenous ligand thereof.
  • a further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a INSP 139 polypeptide according to the present invention and determining the ability of said test compound to modulate the activity of said INSP 139 polypeptide.
  • a further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a INSP 139 gene according to the present invention and determining the ability of said test compound to modulate the expression of said INSPl 39 gene, preferably to stimulate expression thereof.
  • this invention relates to a method of screening, selecting or identifying active compounds, particularly compounds active on multiple sclerosis or related disorders, the method comprising contacting a test compound with a recombinant host cell comprising a reporter construct, said reporter construct comprising a reporter gene under the control of a INSP 139 gene promoter, and selecting the test compounds that modulate (e.g. stimulate or reduce, preferably stimulate) expression of the reporter gene.
  • the polypeptide of the invention can be used to screen libraries of compounds in any of a variety of drug screening techniques. Such compounds may activate (agonise) or inhibit (antagonise) the level of expression of the gene or the activity of the polypeptide of the invention and form a further aspect of the present invention. Preferred compounds are effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
  • Agonist or antagonist compounds may be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, enzymes, receptors or structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et ah, Current Protocols in Immunology l(2):Chapter 5 (1991).
  • Binding to a target gene or polypeptide provides an indication as to the ability of the compound to modulate the activity of said target, and thus to affect a pathway leading to defensin related disorder in a subject.
  • the determination of binding may be performed by various techniques, such as by labelling of the candidate compound, by competition with a labelled reference ligand, etc.
  • the polypeptides may be used in essentially pure form, in suspension, immobilized on a support, or expressed in a membrane (intact cell, membrane preparation, liposome, etc.).
  • Modulation of activity includes, without limitation, stimulation of the surface expression of the INSP139 receptor, modulation of multimerization of said receptor (e.g., the formation of multimeric complexes with other sub-units), etc.
  • the cells used in the assays maybe any recombinant cell (i.e., any cell comprising a recombinant nucleic acid encoding a INSP 139 polypeptide) or any cell that expresses an endogenous INSP 139 polypeptide. Examples of such cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.).
  • E.coli E.coli, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vera cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc).
  • mammalian cell lines e.g., Vera cells, CHO cells, 3T3 cells, COS cells, etc.
  • primary or established mammalian cell cultures e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc).
  • Compounds that are most likely to be good antagonists are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it.
  • Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented.
  • the polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • screening procedures may involve using appropriate cells or cell membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
  • the functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound.
  • Such an assay may assess whether the test compound results in a signal generated by activation of the polypeptide, using an appropriate detection system. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed.
  • a preferred method for identifying an agonist or antagonist compound of a polypeptide of the present invention comprises:
  • a particular example is cotransfecting a construct expressing a polypeptide according to the invention, or a fragment such as the LBD, in fusion with the GAL4 DNA binding domain, into a cell together with a reporter plasmid, an example of which is pFR-Luc (Stratagene Europe, Amsterdam, The Netherlands).
  • This particular plasmid contains a synthetic promoter with five tandem repeats of GAL4 binding sites that control the expression of the luciferase gene. When a potential ligand is added to the cells, it will bind the GAL4-polypeptide fusion and induce transcription of the luciferase gene.
  • a further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises:
  • a method such as FRET detection of ligand bound to the polypeptide in the presence of peptide co-activators might be used.
  • a further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises:
  • the general methods that are described above may further comprise conducting the identification of agonist or antagonist in the presence of labelled or unlabelled ligand for the polypeptide.
  • the method for identifying agonist or antagonist of a polypeptide of the present invention comprises: determining the inhibition of binding of a ligand to cells which express a polypeptide of the invention (and which optionally have a polypeptide of the invention on the surface thereof), or to cell membranes containing such a polypeptide, in the presence of a candidate compound under conditions to permit binding to the polypeptide, and determining the amount of ligand bound to the polypeptide.
  • a compound capable of causing reduction of binding of a ligand is considered to be an agonist or antagonist.
  • the ligand is labelled.
  • a method of screening for a polypeptide antagonist or agonist compound comprises the steps of:
  • step (c) adding a candidate compound to a mixture of labelled ligand and the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium;
  • step (d) measuring the amount of labelled ligand bound to the whole cell or the cell membrane after step (c);
  • step (e) comparing the difference in the labelled ligand bound in step (b) and (d), such that the compound which causes the reduction in binding in step (d) is considered to be an agonist or antagonist.
  • step (c) adding a candidate compound to a mixture of labelled ligand and immobilized polypeptide on the solid support, the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium;
  • step (d) measuring the amount of labelled ligand bound to the immobilized polypeptide or the whole cell or the cell membrane after step (c);
  • step (e) comparing the difference in the labelled ligand bound in step (b) and (d), such that the compound which causes the reduction in binding in step (d) is considered to be an agonist or antagonist.
  • polypeptides may be found to modulate a variety of physiological and pathological processes in a dose-dependent manner in the above-described assays.
  • the "functional equivalents" of the polypeptides of the invention include polypeptides that exhibit any of the same modulatory activities in the above-described assays in a dose-dependent manner.
  • the degree of dose-dependent activity need not be identical to that of the polypeptides of the invention, preferably the "functional equivalents" will exhibit substantially similar dose-dependence in a given activity assay compared to the polypeptides of the invention.
  • simple binding assays may be used, in which the adherence of a test compound to a surface bearing the polypeptide is detected by means of a label directly or indirectly associated with the test compound or in an assay involving competition with a labelled competitor.
  • competitive drug screening assays may be used, in which neutralising antibodies that are capable of binding the polypeptide specifically compete with a test compound for binding, hi this manner, the antibodies can be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide.
  • Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells.
  • an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured.
  • a beta defensin antimicrobial assay is described by Nizet et al. (Nature 2001, 414:454-457).
  • a theta defensin antimicrobial and antiviral assay is described by Cole et al. (Proc. Natl. Acad. Sci. 2002, 99(4):1813-1818).
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564).
  • This method large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed.
  • One way of immobilising the polypeptide is to use non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • the polypeptide of the invention may be used to identify membrane-bound or soluble receptors, through standard receptor binding techniques that are known in the art, such as ligand binding and crosslinking assays in which the polypeptide is labelled with a radioactive isotope, is chemically modified, or is fused to a peptide sequence that facilitates its detection or purification, and incubated with a source of the putative receptor (for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids).
  • a source of the putative receptor for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids.
  • the efficacy of binding may be measured using biophysical techniques such as surface plasmon resonance and spectroscopy.
  • Binding assays may be used for the purification and cloning of the receptor, but may also identify agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its receptor. Standard methods for conducting screening assays are well understood in the art.
  • this invention relates to the use of a INSP 139 polypeptide or fragment thereof, whereby the fragment is preferably a INSP 139 gene-specific fragment, for isolating or generating an agonist or stimulator of the INSPl 39 polypeptide for the treatment of an immune related disorder, wherein said agonist or stimulator is selected from the group consisting of:
  • a specific antibody or fragment thereof including: a) a chimeric, b) a humanized or c) a fully human antibody, as well as;
  • an antibody-mimetic such as a) an anticalin or b) a fibronectin-based binding molecule (e.g. trinectin or adnectin).
  • Anticalins are also known in the art (Vogt et al., 2004). Fibronectin-based binding molecules are described in US6818418 and WO2004029224. Furthermore, the test compound may be of various origin, nature and composition, such as any small molecule, nucleic acid, lipid, peptide, polypeptide including an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain ⁇ e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), etc., in isolated form or in mixture or combinations.
  • an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific
  • the invention also includes a screening kit useful in the methods for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, that are described above.
  • the invention includes the agonists, antagonists, ligands, receptors, substrates and enzymes, and other compounds which modulate the activity or antigenicity of the polypeptide of the invention discovered by the methods that are described above.
  • the various moieties of the invention ⁇ i.e. the polypeptides of the first aspect of the invention, a nucleic acid molecule of the second or third aspect of the invention, a vector of the fourth aspect of the invention, a host cell of the fifth aspect of the invention, a ligand of the sixth aspect of the invention, a compound of the seventh aspect of the invention) may be useful in the therapy or diagnosis of diseases.
  • the moieties of the invention for treating or diagnosing a disease one or more of the following assays may be carried out.
  • test compound refers to the test compound as being a protein/polypeptide
  • test compound a person skilled in the art will readily be able to adapt the following assays so that the other moieties of the invention may also be used as the "test compound”.
  • compositions comprising a polypeptide, nucleic acid, ligand or compound of the invention in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier may be suitable as therapeutic or diagnostic reagents, as vaccines, or as other immunogenic compositions, as outlined in detail below.
  • a composition containing a polypeptide, nucleic acid, ligand or compound [X] is "substantially free of impurities [herein, Y] when at least 85% by weight of the total X+Y in the composition is X.
  • X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95%, 98% or even 99% by weight.
  • compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, ligand, or compound of the invention.
  • therapeutically effective amount refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • an effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.
  • a pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent.
  • a pharmaceutically acceptable carrier for administration of a therapeutic agent.
  • Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions of the invention can be administered directly to the subject.
  • the subjects to be treated can be animals; in particular, human subjects can be treated.
  • compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means.
  • Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention.
  • the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.
  • the compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • One approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • an inhibitor compound as described above
  • a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • antagonists are antibodies.
  • such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously.
  • polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question, may be administered.
  • polypeptide may be administered in the form of fragments that retain the relevant portions.
  • expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as the use of antisense nucleic acid molecules (as described above), either internally generated or separately administered.
  • Modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and introns) of the gene encoding the polypeptide.
  • inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • the complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Such oligonucleotides may be administered or may be generated in situ from expression in vivo.
  • Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al, Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3 ! ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine and butosine, as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine and uridine which are not as easily recognised by endogenous endonucleases.
  • One approach comprises administering to a subject a therapeutically effective amount of a compound that activates the polypeptide, i.e., an agonist as described above, to alleviate the abnormal condition.
  • a therapeutic amount of the polypeptide in combination with a suitable pharmaceutical carrier may be administered to restore the relevant physiological balance of polypeptide.
  • Gene therapy may be employed to effect the endogenous production of the polypeptide by the relevant cells in the subject. Gene therapy is used to treat permanently the inappropriate production of the polypeptide by replacing a defective gene with a corrected therapeutic gene.
  • Gene therapy of the present invention can occur in vivo or ex vivo.
  • Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene and introduction of the genetically altered cells back into the patient.
  • in vivo gene therapy does not require isolation and purification of a patient's cells.
  • the therapeutic gene is typically "packaged" for administration to a patient.
  • Gene delivery vehicles may be non-viral, such as liposomes, or replication-deficient viruses, such as adenovirus as described by Berkner, K.L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992) or adeno-associated virus (AAV) vectors as described by Muzyczka, N., in Curr. Top. Microbiol. Immunol., 158, 97-129 (1992) and U.S. Patent No. 5,252,479.
  • a nucleic acid molecule encoding a polypeptide of the invention may be engineered for expression in a replication-defective retroviral vector.
  • This expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide, such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics (1996), T Strachan and A P Read, BIOS Scientific Publishers Ltd).
  • Another approach is the administration of "naked DNA" in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.
  • the invention provides that they can be used in vaccines to raise antibodies against the disease causing agent.
  • Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat disease after infection).
  • Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with pharmaceutically-acceptable carriers as described above, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Additionally, these carriers may function as immunostimulating agents ("adjuvants").
  • the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and other pathogens.
  • vaccines comprising polypeptides are preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the vaccine formulations of the invention may be presented in unit-dose or multi-dose containers.
  • sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • jet injection see, for example, www.powderject.com
  • jet injection may also be useful in the formulation of vaccine compositions.
  • This invention also relates to the use of nucleic acid molecules according to the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the nucleic acid molecules of the invention which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.
  • Nucleic acid molecules for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et al, Nature, 324, 163-166 (1986); Bej, et al, Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et al, J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291-294 (1990)) prior to analysis.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease.
  • the method may comprise the steps of: a)contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe; b)contacting a control sample with said probe under the same conditions used in step a); c)and detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.
  • a further aspect of the invention comprises a diagnostic method comprising the steps of: a)obtaining a tissue sample from a patient being tested for disease; b)isolating a nucleic acid molecule according to the invention from said tissue sample; and c)diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease.
  • an amplification step for example using PCR, may be included.
  • Deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to labelled RNA of the invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly-matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures.
  • the presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand.
  • Such diagnostics are particularly useful for prenatal and even neonatal testing.
  • Point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by other well-known techniques, such as direct DNA sequencing or single-strand conformational polymorphism, (see Orita et ah, Genomics, 5, 874-879 (1989)).
  • a sequencing primer may be used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotides or by automatic sequencing procedures with fluorescent-tags.
  • Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR.
  • point mutations and other sequence variations, such as polymorphisms can be detected as described above, for example, through the use of allele-specific oligonucleotides for PCR amplification of sequences that differ by single nucleotides.
  • DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et al, Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Sl protection or the chemical cleavage method (see Cotton et ah, Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401).
  • mutations such as microdeletions, aneuploidies, translocations, inversions, can also be detected by in situ analysis (see, for example, Keller et al, DNA Probes, 2nd Ed., Stockton Press, New York, N. Y., USA (1993)), that is, DNA or RNA sequences in cells can be analysed for mutations without need for their isolation and/or immobilisation onto a membrane.
  • Fluorescence in situ hybridization is presently the most commonly applied method and numerous reviews of FISH have appeared (see, for example, Trachuck et al, Science, 250, 559-562 (1990), and Trask et al, Trends, Genet., 7, 149-154 (1991)).
  • an array of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to conduct efficient screening of genetic variants, mutations and polymorphisms.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al, Science (1996), VoI 274, pp 610-613).
  • the array is prepared and used according to the methods described in PCT application WO95/11995 (Chee et al); Lockhart, D. J. et al. (1996) Nat. Biotech.
  • Oligonucleotide pairs may range from two to over one million.
  • the oligomers are synthesized at designated areas on a substrate using a light-directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support, hi another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/25116 (Baldeschweiler et al).
  • a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and over one million which lends itself to the efficient use of commercially-available instrumentation.
  • diseases may be diagnosed by methods comprising determining, from a sample derived from a subject, an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • nucleic acid amplification for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and are discussed in some detail above (including radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays).
  • This aspect of the invention provides a diagnostic method which comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand- polypeptide complex; and (b) detecting said complex.
  • Protocols such as ELISA (as previously described), RIA, and FACS for measuring polypeptide levels may additionally provide a basis for diagnosing altered or abnormal levels of polypeptide expression.
  • Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, such as by photometric means.
  • Antibodies which specifically bind to a polypeptide of the invention may be used for the diagnosis of conditions or diseases characterised by expression of the polypeptide, or in assays to monitor patients being treated with the polypeptides, nucleic acid molecules, ligands and other compounds of the invention.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for the polypeptide include methods that utilise the antibody and a label to detect the polypeptide in human body fluids or extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labelled by joining them, either covalently or non-covalently, with a reporter molecule.
  • a wide variety of reporter molecules known in the art may be used, several of which are described above.
  • Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.
  • a diagnostic kit of the present invention may comprise:
  • a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.
  • the kit may further comprise a third container holding an agent for digesting unhybridised RNA.
  • a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention.
  • a diagnostic kit may comprise one or more antibodies that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.
  • kits will be of use in diagnosing a disease or susceptibility to disease in which members of the defensin family are implicated.
  • diseases may include cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis' sarcoma; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellit
  • the diseases are those in which members of the defensin family are implicated, such as sublethal endotoxaemia, septic shock, microbial infection of the amniotic cavity, Jarish-Herxheimer reaction of relapsing fever, infectious diseases of the central nervous system, acute pancreatitis, ulcerative colitis, empyaema, haemolytic uraemic syndrome, meningococcal disease, gastric infection, pertussis, peritonitis, psoriasis, rheumatoid arthritis, sepsis, asthma, HIV, AIDS, glomerulonephritis, otitis media, paranasal sinusitis, labyrinthitis, meningitis, reproductive health disorders, infertility and male infertility.
  • members of the defensin family are implicated, such as sublethal endotoxaemia, septic shock, microbial infection of the amniotic cavity, Jarish-
  • Figure 1 Top 10 BLASTp results against NCBI non-redundant database using SEQ ID NO: 6 (the INSP139 polypeptide)
  • FIG. 1 Alignment between INSP139 polypeptide sequence (SEQ ID NO:6) and neutrophil beta-defensin 12 (Bos taurus)
  • Figure 5 Nucleotide sequence with translation of INSP 139 PCR product cloned using primers INSP139-CP1 and INSP139-CP2
  • Figure 7 Nucleotide sequence with translation of INSP139-SV1 PCR product cloned using primers INSP139-CP3 and INSP139-CP4
  • Figure 8 Map of pCR4-TOPO-INSP139
  • Figure 9 Map of pDONR 221
  • Figure 10 Map of Expression vector pEAK12d
  • Figure 11 Map of Expression vector pDEST12.2
  • Figure 12 Map of pENTR JNSPl 39-6HIS
  • Figure 20 Amino acid sequence alignment of the canine ⁇ -defensin gene family. Dashes are inserted to optimize the alignment and conserved residues are shaded. Internal exon sequences of CBD105 and CSPAGlIc as well as the last several amino acids of the first exon sequence of CBDl 12 have been deleted to maximise the alignment. Long carboxyl terminal tails of several ⁇ -defensins are not shown for sake of clarity. The letter "X" denotes the presence of a premature stop codon in the pseudogene. First exons for some ⁇ - defensins were not identified. Abbreviations: CBD, canine ⁇ -defensin; CSPAGIl, canine sperm-associated antigen 11 ; P, pseudogene.
  • Figure 21 Amino acid sequence alignment of the human ⁇ -defensin gene family. Dashes are inserted to optimize the alignment and conserved residues are shaded. Internal exon sequences of DEFB105 and SPAGlIc as well as the last several amino acids of the first exon sequence of DEFBl 12 have been deleted to maximise the alignment. Long carboxyl terminal tails of several ⁇ -defensins are not shown for sake of clarity. The letter "X" denotes the presence of a premature stop codon in the pseudogene. First exons for some ⁇ - defensins were not identified. Abbreviations: DEFB, ⁇ -defensin; SPAGIl, sperm- associated antigen 11; P, pseudogene.
  • Figure 22 Amino acid sequence alignment of the mouse ⁇ -defensin gene family. Dashes are inserted to optimize the alignment and conserved residues are shaded. Internal exon sequences o ⁇ Derfol2, Deft>52 and Spagllc have been deleted to maximise the alignment. Long carboxyl terminal tails of several ⁇ -defensins are not shown for sake of clarity. The letter "X” denotes the presence of a premature stop codon in the pseudogene. First exons for some ⁇ -defensins were not identified. Abbreviations: Deft), ⁇ -defensin; p, pseudogene.
  • Figure 23 Genomic organisation of five syntenic ⁇ -defensin gene clusters in human and chimpanzee. The position of each gene is represented by a vertical bar. The direction of transcription is indicated by the triangle. The orthologoud genes are linked across species. The gene mapping was derived from the assembled genomes of human (NCBI Build 35 Version 1) and chimpanzee (NCBI Build 1 Version 1), respectively. It is noted tha chimpanzee DEFB130 and DEFB131 are not conclusively mapped, whereas DEFBIlO and DEFB128 are missing in the current genome assembly. Abbreviations: Tel, telomere; Cen, centromere; P, pseudogene; lle/llc, SPAGl le/SP AGl Ic.
  • the INSP139 polypeptide sequence shown in SEQ ID NO:6, was used as a BLAST query against the NCBI non-redundant sequence database. The top hit is to a Bos taurus gene annotated as a defensin ( Figure 1). INSP 139 aligns to this sequence with a significant E- value (3e "7 ) ( Figure 2), thus indicating a high degree of confidence in the prediction.
  • INSP 139 comprises six conserved cysteine residues that are characteristic of beta-defensin family members (see Figures 18 and 19). These 6 cysteine residues form disulphide bridges, i.e. Cys31-Cys59, Cys38-Cys53 and Cys43 and Cys60. INSP139 comprises 10 positively charged residues and 6 negatively charged residues. This gives an overall positive charge to INSP 139, which is common in many antimicrobial peptides (the theoretical pi of INSP 139 is 8.92).
  • Figure 3 show that INSP 139 is predicted to possess a signal peptide at the start of the protein.
  • the signal peptide cleavage site is thought to be between residues 22 and 23 of the INSP 139 polypeptide sequence (Nielsen, H. et al. 1997, Protein Engineering, 10, 1-6; Nielsen, H., and Krogh, A.: Prediction of signal peptides and signal anchors by a hidden Markov model. In Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (ISMB 6), AAAI Press, Menlo Park, California, pp. 122-130 (1998)).
  • First strand cDNA was prepared from a variety of normal human tissue total RNA samples (Clontech, Stratagene, Ambion, Biochain Institute and in-house preparations) using Superscript II RNase H " Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol. Oligo (dT) 15 primer (l ⁇ l at 500 ⁇ g/ml) (Promega), 2 ⁇ g human total RNA, l ⁇ l 1OmM dNTP mix (1OmM each of dATP, dGTP, dCTP and dTTP at neutral pH) and sterile distilled water to a final volume of 12 ⁇ l were combined in a 1.5ml Eppendorf tube, heated to 65°C for 5 min and then chilled on ice.
  • coli RNase H (Invitrogen) was added and the reaction mixture incubated at 37°C for 20 min. The final 21 ⁇ l reaction mix was diluted by adding 179 ⁇ l sterile water to give a total volume of 200 ⁇ l.
  • cDNA templates used for the amplification of INSP 139 were derived from kidney, liver, lung, small intestine, spleen, colon, testis, skin, pancreas, pituitary gland, salivary gland, adrenal gland, eye, throat, and a universal reference sample often cancerous cell lines.
  • PCR primers Two pairs of PCR primers having a length of between 18 and 25 bases were designed for amplifying the predicted coding sequence of the virtual cDNA using Primer Designer Software (Scientific & Educational Software, PO Box 72045, Durham, NC 27722-2045, USA). PCR primers were optimized to have a Tm close to 55 + 10 0 C and a GC content of 40-60%. Primers were selected which had high selectivity for the target sequence (INSP 139) with little or no none specific priming. The primers were designed to form two nested pairs such that INSP139-CP3/INSP139-CP4 primers were positioned slightly internally to primers INSP139-CP1/INSP139-CP2.
  • Gene-specific cloning primers (INSP139-CP1 and INSP139-CP2, Figure 4, Figure 5, Figure 6, and Table 1) were designed to amplify a cDNA fragment of 366 bp covering the entire of the predicted INSP 139 cds.
  • the primer pair was used with the human cDNA samples listed above as PCR templates. This PCRl was performed in a final volume of 50 ⁇ l containing IX Platinum ® Taq High Fidelity (HiFi) buffer, 2mM MgSO 4 , 200 ⁇ M dNTPs, 0.2 ⁇ M of each cloning primer, 2.5 units of Platinum ® Taq DNA Polymerase High Fidelity (HiFi) (Invitrogen), and approximately 20 ng of template cDNA.
  • HiFi IX Platinum ® Taq High Fidelity
  • PCR2 was performed in a final volume of 50 ⁇ l containing IX Platinum ® Taq HiFi buffer, 2mM MgSO 4 , 200 ⁇ M dNTPs, 0.2 ⁇ M of each cloning primer, and 2.5 units of Platinum ® Taq DNA Polymerase HiFi (Invitrogen). Cycling was performed using an MJ Research DNA Engine, programmed as follows: 94° C, 2 min; 25 cycles of 94°C, 30 sec, 55 0 C, 30 sec, and 68°C, 30 sec; followed by 1 cycle at 68°C for 7 min and a holding cycle at 4°C.
  • PCR products were subcloned into the topoisomerase I modified cloning vector (pCR4-TOPO) using the TA cloning kit purchased from the Invitrogen Corporation using the conditions specified by the manufacturer. Briefly, 4 ⁇ l of gel purified PCR product was incubated for 15 min at room temperature with l ⁇ l of TOPO vector and l ⁇ l salt solution. The reaction mixture was then transformed into E, coli strain TOPlO (Invitrogen) as follows: a 50 ⁇ l aliquot of One Shot TOPlO cells was thawed on ice and 2 ⁇ l of TOPO reaction was added. The mixture was incubated for 15 min on ice and then heat shocked by incubation at 42°C for exactly 30s.
  • PCR reaction products were analyzed on 1% agarose gels in 1 X TAE buffer. Colonies which gave PCR products of approximately the expected molecular weight (366 bp or 267 bp + 105 bp due to the multiple cloning site (MCS)) were grown up overnight at 37°C in 5ml L-Broth (LB) containing ampicillin (lOO ⁇ g /ml), with shaking at 220 rpm.
  • MCS multiple cloning site
  • Miniprep plasmid DNA was prepared from the 5ml culture using a Biorobot 8000 robotic system (Qiagen) or Wizard Plus SV Minipreps kit (Promega cat. no. 1460) according to the manufacturer's instructions. Plasmid DNA was eluted in 80 ⁇ l of sterile water. The DNA concentration was measured using a Spectramax 190 photometer (Molecular Devices). Plasmid DNA (200-500ng) was subjected to DNA sequencing with the T7 and T3 primers using the BigDye Terminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. The primer sequences are shown in Table 1. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.
  • Sequence analysis identified a clone, amplified from eye cDNA using primers IN SP 139- CP1/INSP139-CP2, which matched the expected INSP139 sequence except for a single amino acid substitution in the translated sequence (R21G). As this substitution occurred in the predicted signal peptide and was not predicted to affect cleavage, no subsequent mutagenesis was performed.
  • the sequence of the cloned cDNA fragment is shown in Figure 5.
  • the plasmid map of the cloned PCR product, pCR4-TOPO-INSP139 (plasmid ID 15959), is shown in Figure 6.
  • a second PCR product was identified, which had been amplified from the testis cDNA PCRl reaction products using primers INSP 139- CP3/INSP139-CP4, that was missing the first 21 amino acids (63 bp) of predicted exon 2. This altered the predicted signal peptide cleavage site, which if cleaved generates a mature protein which lacks the first 21 amino acids of the original mature INSP139 prediction.
  • the sequence of the cloned cDNA fragment is shown in Figure 7.
  • the plasmid map of the cloned PCR product called INSP139SV1 ( ⁇ CR4-TOPO-INSP139-SVl) (plasmid ID 15960) is shown in Figure 8.
  • the splice variant lacks a predicted myristoylation site which may alter its functional activity.
  • Example 4 Construction of mammalian cell expression vectors for INSP139 and INSP139- SVl
  • Plasmids 15959 and 15960 were used as PCR templates to generate pEAK12d ( Figures 13 and 16) and pDEST12.2 ( Figures 14 and 17) expression clones containing the INSP139 and INSP139-SV1 ORF sequences respectively, with a 3' sequence encoding a 6HIS tag using the GatewayTM cloning methodology (Invitrogen). 4.1 Generation of Gateway compatible INSP139 and INSP139-SV1 ORF fused to an in frame 6HIS tag sequence.
  • the first stage of the Gateway cloning process involves a two step PCR reaction which generates the ORF of INSP139 and INSP139-SV1 (SEQ ID NO: 9) flanked at the 5' end by an attBl recombination site and Kozak sequence, and flanked at the 3' end by a sequence encoding an in frame 6 histidine (6HIS) tag, a stop codon and the attB2 recombination site (Gateway compatible cDNA).
  • 6HIS in frame 6 histidine
  • the first PCR reaction (in a final volume of 50 ⁇ l) contains respectively: l ⁇ l (40ng) of plasmid 15959 or 15960, 1.5 ⁇ l dNTPs (1OmM), lO ⁇ l of 1OX Pfx polymerase buffer, l ⁇ l MgSO4 (5OmM), 0.5 ⁇ l each of gene specific primer (lOO ⁇ M) (INSP139-EX1 and INSP139-EX2 for INSP139 and INSP139-SV1), and 0.5 ⁇ l Platinum Pfx DNA polymerase (Invitrogen).
  • the PCR reaction was performed using an initial denaturing step of 95°C for 2 min, followed by 12 cycles of 94°C for 15s; 55°C for 30s and 68°C for 2min; and a holding cycle of 4°C.
  • the amplification products were directly purified using the Wizard PCR Preps DNA Purification System (Promega) and recovered in 50 ⁇ l sterile water according to the manufacturer's instructions.
  • the second PCR reaction (in a final volume of 50 ⁇ l) contained lO ⁇ l purified PCR 1 product, 1.5 ⁇ l dNTPs (1OmM), 5 ⁇ l of 1OX Pfx polymerase buffer, l ⁇ l MgSO4 (5OmM), 0.5 ⁇ l of each Gateway conversion primer (lOO ⁇ M) (GCP forward and GCP reverse) and 0.5 ⁇ l of Platinum Pfx DNA polymerase.
  • the conditions for the 2nd PCR reaction were: 95°C for 1 min; 4 cycles of 94°C, 15 sec; 50°C, 30 sec and 68°C for 2 min; 25 cycles of 94°C, 15 sec; 55°C, 30 sec and 68 0 C, 2 min; followed by a holding cycle of 4°C.
  • PCR product was visualized on 0.8 % agarose gel in 1 X TAE buffer (Invitrogen) and the bands migrating at the predicted molecular mass (349 bp for INSP139 and 286bp for INSP139- SVl) were purified from the gel using the Wizard PCR Preps DNA Purification System (Promega) and recovered in 50 ⁇ l sterile water according to the manufacturer's instructions.
  • the second stage of the Gateway cloning process involves subcloning of the Gateway modified PCR products into the Gateway entry vector pDONR221 (Invitrogen, Figure 9) as follows: 5 ⁇ l of purified product from PCR2 were incubated with 1.5 ⁇ l pDONR221 vector (O.l ⁇ g/ ⁇ l), 2 ⁇ l BP buffer and 1.5 ⁇ l of BP clonase enzyme mix (Invitrogen) in a final volume of lO ⁇ l at RT for Ih. The reaction was stopped by addition of proteinase K l ⁇ l (2 ⁇ g/ ⁇ l) and incubated at 37°C for a further 10 min. An aliquot of this reaction (l ⁇ l) was used to transform E.
  • pDONR221 Invitrogen, Figure 9
  • coli DHlOB cells by electroporation as follows: a 25 ⁇ l aliquot of DHlOB electrocompetent cells (Invitrogen) was thawed on ice and l ⁇ l of the BP reaction mix was added. The mixture was transferred to a chilled 0.1cm electroporation cuvette and the cells electroporated using a BioRad Gene-PulserTM according to the manufacturer's recommended protocol. SOC media (0.5ml) which had been pre-warmed to room temperature was added immediately after electroporation. The mixture was transferred to a 15 ml snap-cap tube and incubated, with shaking (220 rpm) for Ih at 37°C. Aliquots of the transformation mixture (lO ⁇ l and 50 ⁇ l) were then plated on L-broth (LB) plates containing kanamycin (40 ⁇ g/ml) and incubated overnight at 37°C.
  • LB L-broth
  • Plasmid mini-prep DNA was prepared from 5ml cultures from 6 of the resultant colonies using a Qiaprep Turbo 9600 robotic system (Qiagen). Plasmid DNA (150-200ng) was subjected to DNA sequencing with 2 IM 13 and M 13 Rev primers using the BigDyeTerminator system (Applied Biosystems cat. no. 4336919) according to the manufacturer's instructions. The primer sequences are shown in Table 1. Sequencing reactions were purified using Montage S ⁇ Q 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.
  • Plasmid eluate (2 ⁇ l or approx. 150ng) from one of the clones which contained the correct sequence (p ⁇ NTR_INSP139-6HIS, plasmid ID 16267, Figure 12 and pENTR_INSP139- SV1-6HIS, plasmid ID 16268, Figure 15 respectively) were then used in recombination reactions containing 1.5 ⁇ l of either pEAK12d vector or pDEST12.2 vector ( Figures 10 & 11) (O.l ⁇ g/ ⁇ l), 2 ⁇ l LR buffer and 1.5 ⁇ l of LR clonase (Invitrogen) in a final volume of lO ⁇ l.
  • SOC media (0.5ml) which had been pre-warmed to room temperature was added immediately after electroporation. The mixture was transferred to a 15ml snap-cap tube and incubated, with shaking (220 rpm) for Ih at 37 ° C. Aliquots of the transformation mixture (lO ⁇ l and 50 ⁇ l) were then plated on L-broth (LB) plates containing ampicillin (lOO ⁇ g/ml) and incubated overnight at 37°C.
  • LB L-broth
  • Plasmid mini-prep DNA was prepared from 5ml cultures from 6 of the resultant colonies subcloned in each vector using a Qiaprep Bio Robot 8000 (Qiagen). Plasmid DNA (200- 500ng) in the pEAK12d vector was subjected to DNA sequencing with pEAK12F and pEAK12R primers as described above. Plasmid DNA (200-500ng) in the pDEST12.2 vector was subjected to DNA sequencing with 21M13 and M13Rev primers as described above. Primer sequences are shown in Table 1.
  • CsCl gradient purified maxi-prep DNA was prepared from a 500 ml culture of the sequence verified clone (pEAK12d_INSP139-6HIS and pEAK12d_INSP139-SVl-6HIS plasmid ID numbers 16272 and 16273, Figures 13 and 16 respectively) using the method described by Sambrook J. et ah, 1989 (in Molecular Cloning, a Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press), Plasmid DNA was resuspended at a concentration of l ⁇ g/ ⁇ l in sterile water (or 1OmM Tris-HCl pH 8.5) and stored at -2O 0 C.
  • Endotoxin-free maxi-prep DNA was prepared from a 500ml culture of the sequence verified clone (pDEST12.2_ INSP139-6HIS and pDEST12.2_INSP139-SVl-6HIS plasmid ID numbers 16278 and 16279, Figures 14 and 17) using the EndoFree Plasmid Mega kit (Qiagen).
  • the presence of the transcript for INSP 139 may be investigated by PCR of cDNA from different human tissues.
  • the INSPl 39 transcript may be present at very low levels in the samples tested. Therefore, extreme care is needed in the design of experiments to establish the presence of a transcript in various human tissues as a small amount of genomic contamination in the RNA preparation will provide a false positive result. Thus, all RNA should be treated with DNAse prior to use for reverse transcription.
  • a control reaction may be set up in which reverse transcription was not undertaken (a -ve RT control). For example, l ⁇ g of total RNA from each tissue may be used to generate cDNA using Multiscript reverse transcriptase (ABI) and random hexamer primers.
  • ABSI Multiscript reverse transcriptase
  • a control reaction is set up in which all the constituents are added except the reverse transcriptase (-ve RT control).
  • PCR reactions are set up for each tissue on the reverse transcribed RNA samples and the minus RT controls.
  • INSP139-specific primers may readily be designed on the basis of the sequence information provided herein. The presence of a product of the correct molecular weight in the reverse transcribed sample together with the absence of a product in the minus RT control may be taken as evidence for the presence of a transcript in that tissue. Any suitable cDNA libraries may be used to screen for the INSP 139 transcripts, not only those generated as described above.
  • tissue distribution pattern of the INSP 139 polypeptides will provide further useful information in relation to the function of those polypeptides.
  • Human Embryonic Kidney 293 cells expressing the Epstein-Barr virus Nuclear Antigen (HEK293-EBNA, Invitrogen) are maintained in suspension in Ex-cell VPRO serum-free medium (seed stock, maintenance medium, JRH).
  • Ex-cell VPRO serum-free medium seed stock, maintenance medium, JRH.
  • cells are seeded in 2x T225 flasks (50ml per flask in DMEM / F12 (1:1) containing 2% FBS seeding medium (JRH) at a density of 2x10 5 cells/ml).
  • plasmid DNA is co-transfected with GFP (fluorescent reporter gene) DNA.
  • GFP fluorescent reporter gene
  • the transfection mix is then added to the 2xT225 flasks and incubated at 37°C (5%CO 2 ) for 6 days. Confirmation of positive transfection may be carried out by qualitative fluorescence examination at day 1 and day 6 (Axiovert 10 Zeiss). On day 6 (harvest day), supernatants from the two flasks are pooled and centrifuged (e.g. 4 0 C, 40Og) and placed into a pot bearing a unique identifier. One aliquot (500 ⁇ l) is kept for QC of the 6His-tagged protein (internal bioprocessing QC).
  • Scale-up batches may be produced by following the protocol called "PEI transfection of suspension cells", referenced BP/PEI/HH/02/04, with PolyEthylenelmine from Polysciences as transfection agent.
  • the culture medium sample containing the recombinant protein with a C-terminal 6His tag is diluted with cold buffer A (5OmM NaH 2 PO 4 ; 60OmM NaCl; 8.7 % (w/v) glycerol, pH 7.5).
  • the sample is filtered then through a sterile filter (Millipore) and kept at 4°C in a sterile square media bottle (Nalgene).
  • the purification is performed at 4°C on the VISION workstation (Applied Biosystems) connected to an automatic sample loader (Labomatic).
  • the purification procedure is composed of two sequential steps, metal affinity chromatography on a Poros 20 MC (Applied Biosystems) column charged with Ni ions (4.6 x 50 mm, 0.83ml), followed by gel filtration on a Sephadex G-25 medium (Amersham Pharmacia) column (1,0 x 10cm).
  • the metal affinity column is regenerated with 30 column volumes of EDTA solution (10OmM EDTA; IM NaCl; pH 8.0), recharged with Ni ions through washing with 15 column volumes of a 10OmM NiSO 4 solution, washed with 10 column volumes of buffer A, followed by 7 column volumes of buffer B (50 mM NaH 2 PO 4 ; 60OmM NaCl; 8.7 % (w/v) glycerol, 40OmM; imidazole, pH 7.5), and finally equilibrated with 15 column volumes of buffer A containing 15mM imidazole.
  • EDTA solution 10OmM EDTA; IM NaCl; pH 8.0
  • the sample is transferred, by the Labomatic sample loader, into a 200ml sample loop and subsequently charged onto the Ni metal affinity column at a flow rate of lOml/min.
  • the column is washed with 12 column volumes of buffer A, followed by 28 column volumes of buffer A containing 2OmM imidazole. During the 2OmM imidazole wash loosely attached contaminating proteins are eluted from the column.
  • the recombinant His-tagged protein is finally eluted with 10 column volumes of buffer B at a flow rate of 2ml/min, and the eluted protein is collected.
  • the Sephadex G-25 gel-filtration column is regenerated with 2ml of buffer D (1.137M NaCl; 2.7mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; pH 7.2), and subsequently equilibrated with 4 column volumes of buffer C (137mM NaCl; 2.7mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; 20% (w/v) glycerol; pH 7.4).
  • buffer D (1.137M NaCl; 2.7mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; pH 7.2
  • buffer C 137mM NaCl; 2.7mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; 20% (w/v) glycerol; pH 7.4
  • the peak fraction eluted from the Ni-column is automatically loaded onto the Sephadex G-25 column through the integrated sample loader on the VISION and the protein is eluted with buffer C at a flow rate of 2 ml/min.
  • the fraction was filtered through a sterile centrifugation filter (Millipore), frozen and stored at -8O 0 C.
  • An aliquot of the sample is analyzed on SDS-PAGE (4-12% NuPAGE gel; Novex) Western blot with anti- His antibodies.
  • the NuPAGE gel may be stained in a 0.1 % Coomassie blue R250 staining solution (30% methanol, 10% acetic acid) at room temperature for Ih and subsequently destained in 20% methanol, 7.5% acetic acid until the background is clear and the protein bands clearly visible.
  • the proteins are electrotransferred from the gel to a nitrocellulose membrane.
  • the membrane is blocked with 5% milk powder in buffer E (137mM NaCl; 2.7mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; 0.1 % Tween 20, pH 7.4) for Ih at room temperature, and subsequently incubated with a mixture of 2 rabbit polyclonal anti-His antibodies (G-18 and H-15, 0.2 ⁇ g/ml each; Santa Cruz) in 2.5% milk powder in buffer E overnight at 4°C.
  • the membrane After a further 1 hour incubation at room temperature, the membrane is washed with buffer E (3 x lOmin), and then incubated with a secondary HRP-conjugated anti-rabbit antibody (DAKO, HRP 0399) diluted 1/3000 in buffer E containing 2.5% milk powder for 2 hours at room temperature. After washing with buffer E (3 x 10 minutes), the membrane is developed with the ECL kit (Amersham Pharmacia) for 1 min. The membrane is subsequently exposed to a Hyperfilm (Amersham Pharmacia), the film developed and the western blot image visually analysed.
  • DAKO secondary HRP-conjugated anti-rabbit antibody
  • the protein concentration may be determined using the BCA protein assay kit (Pierce) with bovine serum albumin as standard.
  • overexpression or knock-down of the expression of the polypeptides in cell lines may be used to determine the effect on transcriptional activation of the host cell genome.
  • Dimerisation partners, co-activators and co-repressors of the INSPl 39 polypeptide may be identified by immunoprecipitation combined with Western blotting and immunoprecipitation combined with mass spectroscopy.
  • the cloned INSP139 sequence contains the mutation R21G (SEQ ID NO: 15). This mutation is in the predicted signal peptide sequence. While this mutation should not theoretically affect the predicted signal peptide cleavage site (between G22 and G23), the charge change induced by this mutation may affect targeting to the secretory pathway. A G21 to R mutation in INSP 139 will therefore be generated to see if there is any effect on the secretion in transfected HEK293 cells. In addition, co-expression of both variants to evaluate if one has an effect on the secretion of the other will be carried out.
  • Fibrosis is characterized by the excessive deposition of extracellular matrix, especially collagen.
  • Stromal cells including fibroblasts, express specific pro- and anti-fibrotic proteins.
  • Keratinocyte growth factor (KGF) is a well- characterized anti-fibrotic molecule.
  • oxidative damage and pro-inflammatory stimuli have been proposed to be among major events leading to myofibroblast phenotype and eventually to fibrosis.
  • NF-kB is a mediator of oxidative stress and inflammatory reactions. Based on fibroblast biology, we have developed four cell-based assays, namely fibroblast proliferation, collagen production, NF-kB activation and KGF production assays.
  • the assay is based on fluorescence enhancement mediated by CyQUANT GR dye bound to cellular nucleic acids and measures the proliferative responses of human skin-derived fibroblasts to novel proteins and small molecules.
  • Fibrosis is characterized by the excessive deposition of extracellular matrix, especially collagen.
  • Over production of type I collagen is the main manifestation of systemic sclerosis.
  • TGF ⁇ is known to up-regulate production of collagen in vitro and in vivo.
  • KGF is an important mediator of stroma-to epithelium interactions in many organs (lung, pancreas, kidney, prostate, mammary, gland, uterus) during normal and pathological growth and development. KGF is specifically produced by stromal cells and its receptor is specifically expressed by epithelial cells. It is proposed that KGF might be an important player during pathophysiological reactions in fibrosis and thus can be used as a marker of these reactions.
  • a KGF ELISA assay has been developed and using human lung-derived fibroblasts it has been shown that the KGF production can be significantly up-regulated by IL- l ⁇ and TNF ⁇ and down-regulated by TGF ⁇ . These cytokines will be used as reference molecules in screening for novel proteins capable to induce KGF production.
  • NF- ⁇ B is a mediator of oxidative stress and inflammatory reactions.
  • Swiss 3T3 fibroblasts were generated with a stably integrated NFKB-SEAP (secreted alkaline phosphatase) construct.
  • NFKB-SEAP secreted alkaline phosphatase
  • the SEAP enzyme is secreted into the culture medium, so samples can be collected at various time points to assay for transcription activity without harvesting cells.
  • the Swiss 3T3 -NFKB-SEAP cell line can be used as a cell-based assay to test novel Functional Genomics proteins and is very promising for testing small molecules, especially those with predicted proVanti-inflammatory activity.
  • CTGF Connective tissue growth factor
  • CTGF a 38-kD cysteine-rich protein, stimulates the production of extracellular matrix elements by fibroblasts.
  • CTGF overexpression has reportedly been found in many fibrotic human tissues, including lung, skin, liver, kidney and blood vessels.
  • TGF ⁇ activates CTGF gene transcription in human lung fibroblasts.
  • a CTGF promoter-reporter was constructed with secreted alkaline phosphatase (SEAP) as a reporter and Swiss 3T3 fibroblasts were generated with a stably integrated CTGF-SEAP construct. Using these fibroblasts it was shown that CTGF promoter is down-regulated by SARP-I, OPG and FSH and up-regulated by TGF ⁇ .
  • SEAP alkaline phosphatase
  • KL-6 originally discovered as a pulmonary adenocarcinoma-related protein and later referred to as MUC-I, is a high-molecular- weight glycoprotein, now classified as Cluster 9 antigen.
  • KL-6 is elevated in both sera and BALF of patients with idiopathic pulmonary fibrosis (IPF) and other lung interstitial diseases, hi lung tissue from patients suffering from IPF, the majority of cells labelled with KL-6 antibodies are regenerating type II pneumocytes.
  • Two peptides were designed to produce polyclonal antibodies against KL-6.
  • KL-6 ELISA can be used to measure KL-6 production by human lung-derived type II pneumocytes.
  • TRAIL has been shown to be one of the cellular ligands for osteoprotegerin (OPG). This assay can be used to measure the biological activity of OPG.
  • RANKL receptor activator of NF-kB ligand
  • RANKL is another ligand for OPG. This assay can also be used to measure the biological activity of OPG.
  • Fas-Ligand-induced T cell death This assay will reveal new modulators of receptor mediated cell death.
  • T cell apoptosis is induced by stimulating Jurkat cells (a human T cell line) with recombinant 6 Histidine-tagged Fas Ligand combined with a monoclonal anti 6-his antibody. Death is quantified by release of LDH, a cytoplasmic enzyme released in the culture medium when cells are dying. The read out is a colorimetric assay read at 490nm.T cells have been shown to be pathogenic in many autoimmune diseases, being able to control antigen-specific T cell death is a therapeutic strategy (e.g. anti-TNF ⁇ treatment in patient with Crohn's disease).
  • Human-MLR proliferation and cytokine secretion.
  • This cell-based assay measures the effects of novel proteins on lymphocyte proliferation and cytokine secretion or inhibition upon stimulation by PBMC from another donor (alloreactivity). These assay address antigen-specific T cell and antigen presenting cell functions, which are crucial cellular responses in any autoimmune diseases.
  • Secreted cytokine IL-2, 4, 5, 10, TNF- ⁇ and IFN- ⁇
  • Mouse-MLR proliferation. This cell-based assay measures the effects of novel proteins on lymphocyte proliferation or inhibition of mouse spleen cells following stimulation by spleen cells from another donor (mouse strain). This cell-based assay measures the effect of novel proteins on T lymphocyte and antigen presenting cell responses and will be used to confirm activity of positives and hits identify in the h-MLR assays. This assay will be use to select proteins that will be tested in murine model of human diseases.
  • Superantigens are strong modulators of the immune system affecting T cells. Superantigens influence immunologically mediated disorders such as IBD, inflammatory skin diseases like atopic dermatitis and psoriasis, hi this cellular assay, we are specifically targeting T lymphocyte activation via the TCR but with different requirements than the T cell response to classical antigens, in particular in respect to co-stimulatory molecules.
  • immunologically mediated disorders such as IBD, inflammatory skin diseases like atopic dermatitis and psoriasis, hi this cellular assay, we are specifically targeting T lymphocyte activation via the TCR but with different requirements than the T cell response to classical antigens, in particular in respect to co-stimulatory molecules.
  • PBMC Human PBMC stimulated with either ConA or PHA.
  • CBA cytokine bead array
  • cytokines can have dual actions, pro or anti-inflammatory, depending of the injury, milieu and cellular target.
  • Any protein with the capability to modulate cytokine secretion may have a therapeutic potential (e.g. decreasing IFN- ⁇ and TNF- ⁇ would be beneficial in ThI -mediated autoimmune disease in contrast decreasing IL-4, IL-5 may be beneficial in Th2-mediated-diseases, inducing IL-10 would interesting in MS and SLE).
  • PBMC Human PBMC stimulated with LPS.
  • This cell-based assay measures the effects of novel proteins on cytokine secretion (IFN- ⁇ , TNF- ⁇ ) induced by LPS acting on monocytes/macrophages and granulocytes.
  • Neutrophils are important in inflammation and autoimmune diseases such as Rheumatoid Arthritis.
  • Leukocyte chemo-attractants such as IL-8 initiate a sequence of adhesive interactions between cells and the micro-vascular endothelium, resulting in activation, adhesion and finally migration of neutrophils.
  • the tissue infiltration of neutrophils depends on a reorganisation of cytoskeleton elements associated with specific changes in cell morphology of these cells.
  • This cell-based assay measures the effect of novel proteins on cytoskeleton reorganization of human neutrophils.
  • This cell-based assay measures the effect of novel proteins on B cell survival.
  • B cell co-stimulation This cell-based assay measures the effect of novel proteins on B cell co-stimulation.
  • THP-I calcium flux The Ca + -fiux in THPl -cell assay measures the effects of novel proteins on their ability to trigger an intracellular calcium release (a generic second messenger events) from the endoplasmic reticulum.
  • Microglia cell proliferation (will be presented to the next IAC).
  • M-CSF is crucial for the final step of maturation of macrophages/microglia and is not replaceable by any other factor.
  • the evaluation of this biological response may represent a way to influence the microglial activity and therefore an opportunity to identify molecules with therapeutic potential fro MS.
  • a cell-based assay was developed to measure the proliferative response of a microglia cell line to M-CSF. The feasibility and the robustness phases showed optimal results. This assay is in 96 well plates; non-radioactive substrate is required, and it is easily automated.

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Abstract

L'invention concerne une nouvelle protéine, appelée INSP 139, identifiée comme étant une protéine sécrétée, et en particulier, comme étant membre de la famille des défensines. L'invention concerne également l'utilisation de cette protéine et d'une séquence d'acides nucléiques du gène codant pour cette protéine dans le diagnostic, la prévention et le traitement de maladies.
PCT/GB2006/002636 2005-07-14 2006-07-14 Proteines de type defensine WO2007007116A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010007165A2 (fr) * 2008-07-18 2010-01-21 Novozymes A/S Traitement de maladies inflammatoires par des bêta-défensines de mammifères
WO2010007166A2 (fr) * 2008-07-18 2010-01-21 Novozymes A/S Traitement de maladies intestinales inflammatoires par des bêta-défensines de mammifères
WO2010007168A3 (fr) * 2008-07-18 2010-06-17 Novozymes Adenium Biotech A/S Traitement de la polyarthrite rhumatoïde avec des bêta défensines de mammifère
EP2279202A1 (fr) * 2008-04-21 2011-02-02 Singapore Health Services Pte Ltd Formes multimeres de peptides antimicrobiens
CN103554244A (zh) * 2013-05-09 2014-02-05 香雪集团(香港)有限公司 具有抑制呼吸道病毒感染的活性的肽及其应用和制备方法
US9217021B2 (en) 2011-07-08 2015-12-22 Defensin Therapeutics Aps Oral treatment of inflammatory bowel disease
US9220264B2 (en) 2008-04-21 2015-12-29 Singapore Health Services Pte Ltd. Multimeric forms of antimicrobial peptides
EP3099706B1 (fr) * 2014-01-29 2018-10-31 BioNTech AG Mimotopes peptidiques de claudine 18.2 et leurs utilisations
CN114891819A (zh) * 2022-06-17 2022-08-12 华南农业大学 一种猪β-防御素129蛋白及其多克隆抗体的制备方法及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002004487A2 (fr) * 2000-07-11 2002-01-17 Ipf Pharmaceuticals Gmbh Procede d'extraction et d'utilisation de nouvelles defensines humaines en tant que proteines biologiquement actives pour traiter des infections et autres pathologies
WO2003024992A2 (fr) * 2001-09-21 2003-03-27 University Of Iowa Research Foundation $g(b)-defensines humaines et murines, et peptides antimicrobiens
WO2004063219A1 (fr) * 2003-01-13 2004-07-29 Ares Trading S.A. Proteines defensines

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002004487A2 (fr) * 2000-07-11 2002-01-17 Ipf Pharmaceuticals Gmbh Procede d'extraction et d'utilisation de nouvelles defensines humaines en tant que proteines biologiquement actives pour traiter des infections et autres pathologies
WO2003024992A2 (fr) * 2001-09-21 2003-03-27 University Of Iowa Research Foundation $g(b)-defensines humaines et murines, et peptides antimicrobiens
WO2004063219A1 (fr) * 2003-01-13 2004-07-29 Ares Trading S.A. Proteines defensines

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
COLE ALEXANDER M ET AL: "Retrocyclin: A primate peptide that protects cells from infection by T- and M-tropic strains of HIV-1", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 99, no. 4, 19 February 2002 (2002-02-19), pages 1813 - 1818, XP002401721, ISSN: 0027-8424 *
DATABASE EMBL [online] 18 April 2006 (2006-04-18), "Beta-defensin 109 precursor (Defensin, beta 109).", XP002401725, retrieved from EBI Database accession no. Q30KR1 *
DATABASE EMBL [online] 18 April 2006 (2006-04-18), "Beta-defensin 109 precursor (Defensin, beta 109).", XP002401726, retrieved from EBI Database accession no. Q30KL7 *
DATABASE EPO Proteins [online] 6 February 2002 (2002-02-06), "Sequence 34 from Patent WO0204487.", retrieved from EBI accession no. EPOP:AX354132 Database accession no. AX354132 *
DATABASE Geneseq [online] 16 July 2003 (2003-07-16), "Human beta-defensin peptide DEFB09.", retrieved from EBI accession no. GSN:ABR43586 Database accession no. ABR43586 *
DATABASE Geneseq [online] 16 July 2003 (2003-07-16), "Human beta-defensin peptide DEFB9 SEQ ID NO:16.", retrieved from EBI accession no. GSN:ABR43511 Database accession no. ABR43511 *
DATABASE Geneseq [online] 28 May 2002 (2002-05-28), "Human beta-defensin hBD-13 peptide fragment #1.", retrieved from EBI accession no. GSN:AAM49591 Database accession no. AAM49591 *
NIZET V ET AL: "Innate antimicrobial peptide protects the skin from invasive bacterial infection", NATURE, NATURE PUBLISHING GROUP, LONDON, GB, vol. 414, no. 6862, 22 November 2001 (2001-11-22), pages 454 - 457, XP002275343, ISSN: 0028-0836 *
PATIL AMAR A ET AL: "Cross-species analysis of the mammalian beta-defensin gene family: presence of syntenic gene clusters and preferential expression in the male reproductive tract", PHYSIOLOGICAL GENOMICS, vol. 23, no. 1, September 2005 (2005-09-01), pages 5 - 17, XP002401720, ISSN: 1094-8341 *
PAZGIER M ET AL: "Human beta-defensins.", CELLULAR AND MOLECULAR LIFE SCIENCES : CMLS. JUN 2006, vol. 63, no. 11, June 2006 (2006-06-01), pages 1294 - 1313, XP002401722, ISSN: 1420-682X *

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US9220264B2 (en) 2008-04-21 2015-12-29 Singapore Health Services Pte Ltd. Multimeric forms of antimicrobial peptides
JP2015145373A (ja) * 2008-04-21 2015-08-13 シンガポール ヘルス サービシーズ ピーティーイー リミテッド 抗菌ペプチド多量体
EP2853539A1 (fr) * 2008-04-21 2015-04-01 Singapore Health Services Pte Ltd Formes multimères de peptides antimicrobiens
US8809262B2 (en) 2008-04-21 2014-08-19 Singapore Health Service Pte Ltd. Multimeric forms of antimicrobial peptides
EP2279202A1 (fr) * 2008-04-21 2011-02-02 Singapore Health Services Pte Ltd Formes multimeres de peptides antimicrobiens
JP2011521902A (ja) * 2008-04-21 2011-07-28 シンガポール ヘルス サービシーズ ピーティーイー リミテッド 抗菌ペプチド多量体
AU2009238701B2 (en) * 2008-04-21 2013-06-20 Agency For Science, Technology And Research Multimeric forms of antimicrobial peptides
CN102159234A (zh) * 2008-07-18 2011-08-17 诺维信阿德宁生物技术公司 使用哺乳动物β防御素的类风湿性关节炎的治疗
US8802621B2 (en) 2008-07-18 2014-08-12 Defensin Therapeutics Aps Treatment of inflammatory bowel diseases with mammal beta defensins
JP2011225596A (ja) * 2008-07-18 2011-11-10 Novozymes Adenium Biotech As 哺乳動物のベータ・ディフェンシンを用いた、炎症性腸疾患の治療
JP2011528333A (ja) * 2008-07-18 2011-11-17 ノボザイムス アデニウム バイオテック アクティーゼルスカブ 哺乳動物のベータ・ディフェンシンを用いた、炎症性腸疾患の治療
JP2011528332A (ja) * 2008-07-18 2011-11-17 ノボザイムス アデニウム バイオテック アクティーゼルスカブ 哺乳動物ベータ・デフェンシンを用いた炎症性疾患の処置
JP2011528334A (ja) * 2008-07-18 2011-11-17 ノボザイムス アデニウム バイオテック アクティーゼルスカブ 哺乳動物のベータ・ディフェンシンを用いた、関節リウマチの治療
EP2399600A1 (fr) * 2008-07-18 2011-12-28 Novozymes Adenium Biotech A/S Traitement de les maladies intestinales inflammatoires par des bêta-défensines de mammifères
US8232242B2 (en) 2008-07-18 2012-07-31 Novozymes Adenium Biotech A/S Treatment of inflammatory bowel diseases with mammal beta defensins
US8232248B2 (en) 2008-07-18 2012-07-31 Novozymes Adenium Biotech A/S Treatment of rheumatoid arthritis with mammal beta defensins
WO2010007165A2 (fr) * 2008-07-18 2010-01-21 Novozymes A/S Traitement de maladies inflammatoires par des bêta-défensines de mammifères
US9279010B2 (en) 2008-07-18 2016-03-08 Defensin Therapeutics Aps Treatment of inflammatory bowel diseases with mammal beta defensins
CN102159232A (zh) * 2008-07-18 2011-08-17 诺维信阿德宁生物技术公司 使用哺乳动物β防御素治疗炎性疾病
WO2010007168A3 (fr) * 2008-07-18 2010-06-17 Novozymes Adenium Biotech A/S Traitement de la polyarthrite rhumatoïde avec des bêta défensines de mammifère
WO2010007166A3 (fr) * 2008-07-18 2010-06-03 Novozymes Adenium Biotech A/S Traitement de maladies intestinales inflammatoires par des bêta-défensines de mammifères
WO2010007165A3 (fr) * 2008-07-18 2010-06-03 Novozymes Adenium Biotech A/S Traitement de maladies inflammatoires par des bêta-défensines de mammifères
WO2010007166A2 (fr) * 2008-07-18 2010-01-21 Novozymes A/S Traitement de maladies intestinales inflammatoires par des bêta-défensines de mammifères
US9217021B2 (en) 2011-07-08 2015-12-22 Defensin Therapeutics Aps Oral treatment of inflammatory bowel disease
US9833495B2 (en) 2011-07-08 2017-12-05 Defensin Therapeutics Aps Oral treatment of inflammatory bowel disease
CN103554244A (zh) * 2013-05-09 2014-02-05 香雪集团(香港)有限公司 具有抑制呼吸道病毒感染的活性的肽及其应用和制备方法
EP3099706B1 (fr) * 2014-01-29 2018-10-31 BioNTech AG Mimotopes peptidiques de claudine 18.2 et leurs utilisations
US10858415B2 (en) 2014-01-29 2020-12-08 Tron—Translationale Onkologie An Der Universitatsmedizin Der Johannes Guttenberg-Universitat Mainz Gemeinnuizige Gmbh Peptide mimotopes of claudin 18.2 and uses thereof
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